Pesticides in the Modern World - Pests Control and ...

Pesticides in the Modern World - Pests Control and Pesticides Exposure and Toxicity Assessment

Edited by: Margarita Stoytcheva ISBN 978-953-307-457-3, Hard cover, 614 pages Publisher: InTech Publication date: October 2011 Subject: Biochemistry

The present book is a collection of selected original research articles and reviews providing adequate and up-to-date information related to pesticides control, assessment, and toxicity. The first section covers a large spectrum of issues associated with the ecological, molecular, and biotechnological approaches to the understanding of the biological control, the mechanism of the biocontrol agents action, and the related effects. Second section provides recent information on biomarkers currently used to evaluate pesticide exposure, effects, and genetic susceptibility of a number of organisms. Some antioxidant enzymes and vitamins as biochemical markers for pesticide toxicity are examined. The inhibition of the cholinesterases as a specific biomarker for organophosphate and carbamate pesticides is commented, too. The third book section addresses to a variety of pesticides toxic effects and related issues including: the molecular mechanisms involved in pesticides-induced toxicity, fish histopathological, physiological, and DNA changes provoked by pesticides exposure, anticoagulant rodenticides mode of action, the potential of the cholinesterase inhibiting organophosphorus and carbamate pesticides, the effects of pesticides on bumblebee, spiders and scorpions, the metabolic fate of the pesticide-derived aromatic amines, etc. Available from: http://www.intechopen.com/books/show/title/pesticides-in-the-modern-world-pests-control-andpesticides-exposure-and-toxicity-assessment

PESTICIDES IN THE MODERN WORLD – PESTS CONTROL AND PESTICIDES EXPOSURE AND TOXICITY ASSESSMENT Edited by Margarita Stoytcheva

Pesticides in the Modern World – Pests Control and Pesticides Exposure and Toxicity Assessment Edited by Margarita Stoytcheva

Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access articles distributed under the Creative Commons Non Commercial Share Alike Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published articles. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Sandra Bakic Technical Editor Teodora Smiljanic Cover Designer Jan Hyrat Image Copyright luri, 2010. Used under license from Shutterstock.com First published September, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected]

Pesticides in the Modern World – Pests Control and Pesticides Exposure and Toxicity Assessment, Edited by Margarita Stoytcheva p. cm. ISBN 978-953-307-457-3

free online editions of InTech Books and Journals can be found at www.intechopen.com

Contents Preface IX Part 1 Chapter 1

Biocontrol of Pests

1

Using Ecological Knowledge and Molecular Tools to Develop Effective and Safe Biocontrol Strategies Martina Köberl, Elshahat M. Ramadan, Bettina Roßmann, Charles Staver, Michael Fürnkranz, Birgit Lukesch, Martin Grube and Gabriele Berg

3

Chapter 2

Development of RNAi in Insects and RNAi-Based Pest Control 27 Guang Yang, Minsheng You, Liette Vasseur, Yiying Zhao and Chunhui Liu

Chapter 3

Evaluation of Plant Extracts on Mortality and Tunneling Activities of Subterranean Termites in Pakistan 39 Sohail Ahmed, Mazhar Iqbal Zafar, Abid Hussain, Muhammad Asam Riaz and Muhammad Shahid

Chapter 4

Botanical Insecticides and Their Effects on Insect Biochemistry and Immunity 55 Arash Zibaee

Chapter 5

The Production, Separation and Stability of Pyoluteorin: A Biological Pesticide 69 Wei Wang, Hui Dong, Jingfang Zhang, Yuquan Xu and Xuehong Zhang

Chapter 6

Using the Bio-Insecticide Bacillus Thuringiensis Israelensis in Mosquito Control 93 Després Laurence, Lagneau Christophe and Frutos Roger

Chapter 7

Screening of Biocontrol Agents Against Rhizoctonia solani Causing Web Blight Disease of Groundnut (Arachis hypogaea L.) 127 Sevugaperumal Ganesan and Rajagobal Sekar

VI

Contents

Chapter 8

Part 2

Optimization of the Strategy for Recombinant Baculovirus Infection of Suspended Insect Cells 141 Guohong Zhou, Youhong Zhang and Yujie Ke Pesticides Biomarkers

159

Chapter 9

Pesticide Biomarkers 161 Rojas-García AE, Medina-Díaz IM, Robledo-Marenco ML, Barrón-Vivanco BS and Pérez-Herrera N

Chapter 10

Biological Markers of Human Exposure to Pesticides Manel Araoud

Chapter 11

Pesticide Biomarkers in Terrestrial Invertebrates Juan C. Sanchez-Hernandez

Chapter 12

Biomarkers of Pesticide - Contaminated Environment O. Otitoju and I.N.E. Onwurah

Chapter 13

Chapter 14

Part 3

191

213

241

Fish Cholinesterases as Biomarkers of Organophosphorus and Carbamate Pesticides Caio Rodrigo Dias Assis, Ranilson Souza Bezerra and Luiz Bezerra Carvalho Jr

253

The Effects of Pesticides on Dictyostelium Cholinesterase, from Basic to Applied Research 279 Andrea Amaroli Pesticides Toxicity 295

Chapter 15

Molecular Mechanisms of Pesticide Toxicity 297 Olfa Tebourbi, Mohsen Sakly and Khémais Ben Rhouma

Chapter 16

Structural and Dynamic Basis of Serine Proteases from Nematophagous Fungi for Cuticle Degradation 333 Shu-Qun Liu, Lian-Ming Liang, Tao Yan, Li-Quan Yang, Xing-Lai Ji, Jin-Kui Yang, Yun-Xin Fu and Ke-Qin Zhang

Chapter 17

The Effects of Pyrethroid and Triazine Pesticides on Fish Physiology 377 Josef Velisek, Alzbeta Stara and Zdenka Svobodova

Chapter 18

Genotoxicity Testing in Pesticide Safety Evaluation 403 Gargee Mohanty, Jyotirmayee Mohanty, Shubha Dipta Jena and S.K. Dutta

Contents

Chapter 19

Gene Expressions of the Dhb, Vtg, Arnt, CYP4, CYP314 in Daphnia magna Induced by Toxicity of Glyphosate and Methidathion Pesticides 427 Thai-Hoang Le, Jiho Min, Sung-Kyu Lee and Yang-Hoon Kim

Chapter 20

Challenges of Anticoagulant Rodenticides: Resistance and Ecotoxicology 441 Philippe Berny

Chapter 21

Morphogenetic Activities of Bendiocarb as Cholinesterase Inhibitor on Development of the Chick Embryo 469 Eva Petrovova, Lenka Luptakova, David Mazensky, Jan Danko and David Sedmera

Chapter 22

Age-Related Differences in Acetylcholinesterase Inhibition Produced by Organophosphorus and N-Methyl Carbamate Pesticides 495 Virginia C Moser

Chapter 23

Side-Effects of Pesticides on the Pollinator Bombus: An Overview 507 Veerle Mommaerts and Guy Smagghe

Chapter 24

Chemical Control of Spiders and Scorpions in Urban Areas 553 Eduardo Novaes Ramires, Mario Antonio Navarro-Silva and Francisco de Assis Marques

Chapter 25

Pesticide-Derived Aromatic Amines and Their Biotransformation 601 Jean-Marie Dupret, Julien Dairou, Florent Busi, Philippe Silar, Marta Martins, Christian Mougin, Fernando Rodrigues-Lima and Angelique Cocaign

VII

Preface Volume 5 of the book series “Pesticides in the Modern World” is a collection of selected original research articles and reviews dedicated to the following main topics: biocontrol of pests, pesticides biomarkers, and pesticides toxicity. The first section (Chapters 1-8) covers a large spectrum of issues associated with the ecological, molecular, and biotechnological approaches to the understanding of the biological control, the mechanism of the biocontrol agents action, and the related effects. Three examples, given in Chapter 1, illustrate the development of effective and safe biocontrol strategies, namely: control of soil-borne pathogens on medical plants under organic conditions in Egypt, control of fungal pathogens in banana in Uganda, and control of a multi-species disease in the Styrian oilseed pumpkin. In Chapter 2 is summarized the current knowledge on RNA interference research on insects, and the potential application of RNAi in integrated pest management. Using of plant extracts for termites repelling in Pakistan is the subject of Chapter 3. The effects of botanical insecticides on digestive and on detoxifying enzymes, as well as on the immunological system of insects are discussed in Chapter 4. Useful data for the further development of Pseudomonas spp. cultivation process in the large-scale production and the commercial use of the biological pesticide pyoluteorin are provided in Chapter 5. The action of Bacillus thuringiensis israelensis toxins after ingestion by mosquito larvae and the diversity of mechanisms involved in mosquito resistance are described in Chapter 6. The results of the screening of biocontrol agents against Rhizoctonia solani causing web blight diseases of groundnut are reported in Chapter 7. In Chapter 8 are presented experimental data helpful for the optimization of the process of development of the insect-specific baculoviruses, used as biological insecticides. The second book section (Chapters 9-14) provides recent information on biomarkers research for pesticides exposure assessment. The biomarkers currently used to evaluate pesticide exposure, effects, and the genetic susceptibility of aquatic organisms, terrestrial invertebrates and human populations are revised in Chapters 911. Some antioxidant enzymes and vitamins as biochemical markers for pesticide toxicity are examined in Chapter 12. The inhibition of the cholinesterases as a specific biomarker for organophosphate and carbamate pesticides is commented in Chapters 13 and 14.

X

Preface

The third book section addresses a variety of pesticides toxic effects and related issues. Chapter 15 is intended to summarize the increasing data regarding the molecular mechanisms involved in pesticides-induced toxicity, with relevance to the progression of the most frequent diseases. Several three-dimensional structural models of cuticledegrading serine proteases secreted by nematophagous fungi, helpful for exploiting these enzymes as effective bio-control agents are described in Chapter 16. Investigations on fish histopathological, physiological, and DNA changes induced by pesticides exposure are reported in Chapters 17 and 18, thus contributing to the understanding of the toxicological risks caused by pesticides to ecosystems. Data presented in Chapter 19 demonstrate the hazardous effects of the pesticides glyphosate and methidathion on D. magna by studying the changes in the gene expressions of five stress responsive genes, including Dhb, Arnt, Vtg, CYP4, and CYP314. Chapter 20 provides details on anticoagulant rodenticides mode of action and on the strategies for evaluating and managing pesticides resistance in rodents. The potential of the cholinesterase inhibiting organophosphorus and carbamate pesticides is discussed in Chapters 21 and 22. A comprehensive overview of the side-effects of pesticides including discussion on the testing strategies employed to evaluate pesticide compatibility on bumblebees is provided in Chapter 23. The effects of pesticides on spiders and scorpions, the techniques applied for chemical control of arachnids, and the biology of these arthropods are reviewed in Chapter 24. The metabolic fate of xenobiotics such as pesticide-derived aromatic amines and the strategies for bioremediation of contaminated soils are discussed in Chapter 25. The adequate and up-to-date information related to pesticides control, assessment, and toxicity provided in this book should be of interest for specialists, involved in pest control decisions. Thanks are extended to each of the authors for their efforts in contributing the series “Pesticides in the Modern World”.

Margarita Stoytcheva Mexicali, Baja California Mexico

Part 1 Biocontrol of Pests

1 Using Ecological Knowledge and Molecular Tools to Develop Effective and Safe Biocontrol Strategies *Martina

Köberl1, Elshahat M. Ramadan2, Bettina Charles Staver3, Michael Fürnkranz1, Birgit Lukesch1, Martin Grube4 and Gabriele Berg1 Roßmann1,

1. Introduction Today’s farming systems undermine the well-being of communities in many ways: farming has destroyed huge regions of natural habitats, which also implies a loss of species and their ecosystem services (Sachs et al., 2010). Plant protection measures also causes problems for human health (Horrigan et al., 2002), and agriculture is responsible for about 30% of greenhouse-gas-emission (IPCC, 2007). Furthermore, emerging, re-emerging and endemic plant pathogens continue to challenge our ability to safeguard plant growth and health worldwide (Miller et al., 2009). Therefore, one of the major challenges for the 21st century will be an environmentally sound and sustainable crop production. Microbial inoculants containing microorganisms with beneficial plant-microbe interactions have a great potential to contribute to this objective (Berg, 2009; Bhattacharjee et al., 2008). Over the past 150 years, research has demonstrated repeatedly that bacteria and fungi have an intimate interaction with their host plants and are able to promote plant growth as well as to suppress plant pathogens (Compant et al., 2005; Lugtenberg & Kamilova, 2009; Weller et al., 2002; Weller, 2007; Whipps, 2001). All plant-associated microenvironments, especially the rhizosphere, are colonized in high abundances by antagonistic microbes (Berg et al., 2005a). Between 1 and 35% of the microbial inhabitants showed antagonistic capacity to inhibit the growth of pathogens in vitro (Berg et al., 2002, 2006). The proportion of isolates, which express plant growth promoting traits is much higher in general, and was found up to 2/3 of the cultivable population (Cattelan et al., 1999; Fürnkranz et al., 2009; Lottmann et al., 1999). Diverse microbial inoculants, which were selected from this promising indigenous potential, are already on the market. In recent years, the popularity of microbial inoculants has increased substantially, as extensive and systematic research has enhanced their effectiveness and consistency (Berg, 2009). New molecular and microscopic techniques are one reason for progress in biocontrol research. These techniques have enhanced our understanding about the plant and especially Graz University of Technology, Institute for Environmental Biotechnology, Austria SEKEM and Heliopolis University, Faculty of Agriculture, Cairo, Egypt 3Bioversity International, Banana and Plantain Section, Montpellier, France 4University of Graz, Institute of Plant Sciences, Austria* 1 2

4

Pesticides in the Modern World – Pests Control and Pesticides Exposure and Toxicity Assessment

the rhizosphere as a microbial ecosystem and resulted into more effective screening strategies for bioactive microbes. In this chapter we will discuss these points first in general and in a second part with three representative examples.

2. Molecular and microscopic tools in biocontrol research Molecular and microscopic tools can be used to study the ecology of single plant growth promoting rhizobacteria (PGPR) or biological control agent (BCA) strains or to analyse the structure and function of the target microbial community. In a first step we will analyse the use of methods for single strains (Table 1). Here, molecular fingerprints using repetitive elements in the genome (Rademaker & de Bruijn, 1997) can be used at several levels of biocontrol research. While the functions of many of these repetitive sequence elements are still unknown, they have proven to be useful as the basis of several powerful tools for use in microbial ecology. The repetitive, sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or ‘fingerprints’ of individual microbial strains (Ishii & Sadowsky, 2009). In screening strategies, these fingerprints can be applied to differentiate strains at population level and to select only unique isolates (Berg et al., 2006; Faltin et al., 2004). In a later stage, these highly reproducible fingerprints can be used for identity check and quality control. Genome sequencing also offers a tool to study PGPRs in great detail. Strains of Pseudomonas fluorescens, one of the dominant and cosmopolitan plant-associated species (Weller, 2007), were the first sequenced strains (Paulsen et al., 2005). Genomic information allowed the analysis of the mode of action, detailed investigations of interactions as well as optimisation of fermentation and formulation processes (rev. in Gross & Loper, 2009). De Bruijn et al. (2007) used genome mining to discover unknown gene clusters and traits that are highly relevant in the life style of P. fluorescens SBW25. Proteomic and transcriptomic studies are interesting to study the function of BCAs. For example, Garbeva et al. (2011) studied transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors (Bacillus, Brevundimonas and Pedobacter), which demonstrated that Pf0-1 shows a species-specific response to bacterial competitors. In another transcriptomic study published by Hassan et al. (2010), a whole genome oligonucleotide microarray was developed for P. fluorescens Pf-5 and used to assess the consequences of a gacA mutation: GacA significantly influenced transcript levels of 10% of the 6147 annotated genes in the Pf-5 genome including genes involved in the production of hydrogen cyanide, pyoluteorin and the extracellular protease. Transcriptomic studies can also lead to new insights into plant responses on BCAs: Pseudomonas-primed barley genes indicated that, as is the case in dicots, jasmonic acid plays a role in host responses (Petti et al., 2010). A new tool is metabolomics, which allow the analysis of metabolites in situ. This is not only a technique to answer questions about the activity ad planta, it is also important for registration procedures, which are still a high hurdle on the way to the market. Frimmersdorf et al. (2010) used a metabolomic approach to show how Pseudomonas aeruginosa adapts to various environments. In addition, analysis of the mobilome of strains can result in interesting findings for biocontrol research as shown for P. fluorescens Pf-5 by Mavrodi et al. (2009), in which mobile genetic elements contain determinants that contribute to Pf-5's ability to adapt to changing environmental conditions and/or colonize new ecological niches. Studying the colonisation of plants has been greatly facilitated by the application of fluorescent proteins which are used as vital markers and reporter genes (rev. in Bloemberg, 2007). These new insights have changed our understanding about

Using Ecological Knowledge and Molecular Tools to Develop Effective and Safe Biocontrol Strategies

5

colonisation; many of the strains analysed showed an endophytic life style (Chin-A-Woeng et al., 1997; Zachow et al., 2010), and the “root shield”, which was hypothesized in former times, was rarely found in contrast to single cells and micro-colonies. Raman-FISH combines stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function (Huang et al., 2007a). This potential has been demonstrated through the discriminant functional analysis of Raman spectral profiles (RSP) obtained from the soil and plant-associated bacterium P. fluorescens SBW25; results suggests that SBW25 growth in the phytosphere is generally neither carbon-catabolite-repressed nor carbonlimited (Huang et al., 2007b). Molecular tools were also used to analyse target habitats of biocontrol studies (Table 1). Cultivation-based methods to analyse plant-associated bacteria only address the culturable fraction, which are thought to represent only a small proportion (0.1 to 10%) of the total bacteria present in soil and in the rhizosphere (Amann et al., 1995). The analysis of nucleic acids directly extracted from plant microenvironments opened the chance to study a much broader spectrum of microbes (Table 1). Most frequently ribosomal RNA gene fragments are amplified from total community DNA and subsequently analysed by fingerprinting techniques: Terminal restriction fragment length polymorphism (T-RFLP), single-strand conformation polymorphism (SSCP), denaturing/temperature gradient gel electrophoresis (D/TGGE) using universal/specific primers (Schwieger & Tebbe, 1998; Smalla et al., 2007). Application of these fingerprinting techniques resulted in important findings such as plantspecific microbial communities (Smalla et al., 2001), the impact of cultivars on microbial communities (Milling et al., 2004) or the structure of endophytic communities (Rasche et al., 2006). Fingerprinting techniques are often used to analyse the structure of plant-associated communities and can also be used to study functional aspects. For example, Briones et al. (2002) found cultivar-specific differences for ammonia-oxidizing bacteria (AOB) in rice rhizospheres by a multiphasic approach including DGGE of the amoA gene, analysis of libraries of cloned amoA, fluorescently tagged oligonucleotide probes targeting 16S rRNA of

Objective/Level

Isolates: BCAs and pathogens

Molecular fingerprints

Rep-PCR (BOX)

Genomic information

Genome sequencing Transcriptomics (RNAbased) Proteomics (Protein-based) Metabolome Mobilome GFP/DsRed labelled strains, CLSM Raman spectroscopy and fluorescence in situ hybridization (FISH)

Functions Functional diversity Bioactive compounds Adaptation/evolution

Visualisation/activity

Microbial communities T-RFLP, SSCP, D/TGGE using universal/specific primers Metagenome Metatranscriptome Metaproteome Metabolome Metamobilome

FISH-CLSM

Table 1. Molecular and microscopic tools in biocontrol research.

6


AOBs as well as metabolism rates obtained by the 15N dilution technique. Other techniques have a great impact on our functional understanding; this was shown for example for transcriptome profiling (Mark et al., 2005; Yuan et al., 2008), microarrays (Sanguin et al., 2006; Weinert et al., 2011) in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression (Rediers et al., 2005), new methods for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions (de Werra et al., 2008), barcode pyrosequencing (Gomes et al., 2010), and ultra deep sequencing (Velicer et al., 2006). Stable isotope probing (SIP) used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species resulted in plant species specific patterns (Haichar et al., 2008). Metagenomic approaches have been established to analyse the plant-soil interface (Erkel et al., 2006; rev. in Leveau, 2007).

3. Using ecological knowledge to screen and evaluate biocontrol agents The advanced techniques discussed above should be integrated into strategies to screen and evaluate biocontrol agents (Fig. 1). Of primary importance is the life cycle of the pathogen. This can result in new targets for biocontrol; one example is the impact of zoospores on pathogenic oomycetes, which are primary targets for suppression (de Bruijn et al., 2007; Raaijmakers et al., 2010). Furthermore, it is also important to understand the target microenvironment of plants. Plant specificity is one critical point but also knowledge about the structure and function of the microbial communities. There are strategies to select BCAs from the indigenous antagonistic potential as well as to use ubiquitous, cosmopolitan BCAs (Zachow et al., 2010). If a BCA is selected, an evaluation strategy is needed to assess their potential for commercialization.

Fig. 1. Integration of ecological knowledge into screening and evaluation strategies. Knowledge about the effect of BCAs under greenhouse and field conditions presents the basis for this evaluation. However, often inconsistent effects make the decision difficult. Detailed analyses of plant-microbe and pathogen-microbe interactions under different environmental conditions can help to optimize the biocontrol effect under practical conditions. Another aspect, which should be integrated in an early phase of evaluation, is


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biosafety. Many BCAs fail here due to problems with human or environmental health. Due to the fact that the whole program to investigate toxicology is time-consuming and expensive, alternative test systems should be used, e.g. the Caenorhabditis elegans assay (Zachow et al., 2009) or Duckweed (Lemna minor) as a model plant system for the study of human microbial pathogenesis (Zhang et al., 2010).

4. Examples for screening and evaluation strategies 4.1 Strategy to control soil-borne pathogens on medical plants under organic conditions in Egypt On the SEKEM farms in Egypt desert land was converted into arable land, and biodynamic agriculture is operated for over 30 years now (www.sekem.com). Today SEKEM is carrying out organic agriculture on more than 4100 hectares and has the largest market for organic products outside Europe and North America. They produce organic foods, spices, tea, cotton textiles and natural remedies. However, the cultivation especially of medical plants is more and more affected by soil-borne phytopathogens, which lead to significant yield losses. The objective of our study was to develop a specific biocontrol strategy for desert farming. An important factor was to find out, whether and how the highly specialized natural microbial communities of the desert soil are affected by agriculture and watering. To examine the impact of organic agriculture on bacterial diversity and community compositions in desert soil, soil from a SEKEM farm in comparison to the surrounding desert soil were assessed by a pyrosequencing-based analysis of partial 16S rRNA gene sequences. When appropriate primers are chosen, in a pyrosequencing analysis with short reads the microbial diversity is represented almost as reliably as with near-full-length sequences (Will et al., 2010). Fragments encompassing the V4-V5 region of the 16S rRNA gene provide estimates comparable to those obtained with the nearly complete fragment (Youssef et al., 2009). In desert soil 19244 and in agricultural soil 33384 quality sequences with a read length of ≥ 150 bp were recovered. Using different data bases, 83.0% of all quality sequences could be classified below the domain level, in the range of the percentage of classified 16S rRNA gene sequences of other pyrosequencing-based studies (Lauber et al., 2009; Lazarevic et al., 2009; Will et al., 2010). The computed Shannon indices of diversity (H’) (Shannon, 1997) were much higher for agricultural soil than for desert soil (H’ at a dissimilarity level of 20%: SEKEM soil 4.29; desert soil 3.54); this indicates a higher bacterial diversity in soil due the agricultural use of the desert. A comparison of rarefaction analyses with the number of operational taxonomic units (OTUs) estimated by the Chao1 richness estimator (Chao & Bunge, 2002; Will et al., 2010) revealed that at this genetic distance the surveying effort in both soils covered almost the full extent (over 97% in both soils) of taxonomic diversity. This was also shown by a clear saturation of both curves in the rarefaction analysis (data not shown). The 43673 classifiable sequences obtained from both soil types together were affiliated with 18 different phyla. Dominant groups were especially Proteobacteria (30.2%), Firmicutes (27.3%) and Actinobacteria (10.5%). These dominant phyla were present in both soils. In detail, Firmicutes were highly enriched in agricultural soil (from 11.3% in desert soil to 36.6% in SEKEM soil), Proteobacteria (46.0% in desert soil and 21.0% in SEKEM soil) and Actinobacteria (20.7% in desert soil and 4.6% in SEKEM soil) occurred in SEKEM in lower abundances than in the surrounding desert. In addition, in both soils Bacteroidetes (4.6% and 5.3%) and Gemmatimonadetes (1.4% and 1.9%) were

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present. Whereas Acidobacteria (7.9%) and Planctomycetes (1.1%) were only present in the agricultural soil, Deinococcus-Thermus (1.1%) was only detectable in the desert sand. These abundances of the phyla are coextensive with results from previously reported metaanalysis of bacterial community composition in soils and, despite the specific soil type of the desert, the composition covers rather well with studies of completely different soils (Hansel et al., 2008; Janssen, 2006; Lauber et al, 2009; Will et al., 2010). However, greatly different from all reported studies was the high abundance of Firmicutes. Janssen (2006) reported them to contribute only a mean of 2% (range 0 – 8%) in the total bacterial soil community. Most of the Firmicutes sequences were classified as belonging to the genus Bacillus; in the agricultural soil also the phylogenetically related genus Paenibacillus was found (5% of classified Firmicutes). In desert soil, Ochrobactrum was the most abundant genus within the (Alpha-)Proteobacteria (79% of classified Proteobacteria) and Rhodococcus among the Actinobacteria (90% of classified Actinobacteria). The Acidobacteria in the agricultural soil are affiliated only with subdivision 6. Additionally to the pyrosequencing analysis, the composition of the bacterial as well as fungal community in the two different soil types was investigated by SSCP analysis of rRNA gene fragments (Bassam et al., 1991; Schwieger & Tebbe, 1998). Furthermore, the composition of the microbial community in rhizosphere and endorhiza of three different species of medical plants (Matricaria chamomilla L., Calendula officinalis L. and Solanum distichum Schumach. & Thonn.) grown under organic conditions on SEKEM farms were examined. According to the cluster analysis prepared on the basis of SSCP community fingerprints, the agricultural soil in bacterial as well as in fungal community composition strongly differed from the desert soil. As shown in the pyrosequencing analysis, in comparison to the desert in soil of the SEKEM farm an impressive diversity of bacteria, expressed as various bands in the gel, was found (data not shown). In the bacterial community of the desert soil, two dominant bacterial bands could be detected, which were also visible in all samples from the endorhiza of all three investigated medical plants. This shows that bacteria are taken up by the plants from the soil, and that soil is the main reservoir for biological control agents. The two dominant bands were identified by partial 16S rRNA gene sequence analysis as Ochrobactrum sp. (closest database match O. grignonense) and Rhodococcus sp. (closest database match R. erythropolis). Further, nearly in all samples Bacillus sp. was found (closest database match B. subtilis). By SSCP analysis and also by the pyrosequencing approach, Ochrobactrum and Rhodococcus could be detected as dominant bacteria. However, both genera include opportunistic human pathogens (O. anthropi, R. equi). Several studies provided evidence that similar or even identical functions are responsible for beneficial interactions with plants and virulence in humans (Berg et al., 2011). For Ochrobactrum was already detected the production of plant growth hormones and siderophores and also an antifungal activity towards several phytopathogens was described (Chakraborty et al., 2009). Ochrobactrum was found in diverse environmental niches, like rhizosphere, soil, sediments and activated sludge (Berg et al., 2005b). Rhodococcus could also be found in a broad range of environments, including soil, water and eukaryotic cells. This genus includes also a phytopathogenic species causing leafy gall formation on a wide range of host plants, R. fascians (Goethals et al., 2001). The fungal community fingerprints included a quite high diversity in all microenvironments. As an example, SSCP profiles of fungal communities in rhizosphere and endorhiza are shown in Figure 2. A dominant band, which was found nearly in all samples, was identified as


9

Fig. 2. SSCP profiles of the fungal communities in rhizosphere and endorhiza of the medical plants. Four independent replicates per plant and microenvironment were loaded onto the gel. Std.: 1 kb DNA ladder. Verticillium dahliae, which is one of the mainly occurring soil-borne phytopathogens on the SEKEM farms. In general, mainly potential plant pathogens were found within the fungal communities. The obligate root-infecting pathogen Olpidium, belonging to the fungal phylum Chytridiomycota, was found especially in the rhizosphere and endorhiza of Matricaria chamomilla. Alternaria and Acremonium were found primarily in the rhizosphere samples. According to the generated dendrograms, a clear plant specificity of the bacterial and fungal communities in the rhizosphere as well as in the endorhiza was found (Fig. 3). Furthermore, microenvironment-specific SSCP patterns of the bacterial and the fungal communities were detected (data not shown). There were significant differences between the rhizosphere and the endorhiza of the medical plants. In general, samples from the rhizosphere generated more bands than samples from the endorhiza of the medical plants, which indicate that a sub-set of rhizobacteria was able to invade the root. The major problems in the cultivation of plants on SEKEM farms are caused by the soilborne pathogenic fungi Verticillium dahliae Kleb., Rhizoctonia solani J.G. Kühn and Fusarium culmorum (Wm.G. Sm.) Sacc. as well as by the soil-borne pathogenic bacterium Ralstonia solanacearum. Although grown in organic agriculture, which aims to minimize the impact on the environment by practices such as crop rotation, using pathogen resistant cultivars, and the use of organic manure (compost) instead of synthetic fertilizers (Schmid et al., 2011), they have an increasing importance. One reason is an intensive growing of a limited number


10

A

B

Fig. 3. UPGMA dendrograms of bacterial (A) and fungal (B) communities in rhizosphere and endorhiza of the medical plants. The dendrograms were generated from the SSCP community profiles with GelCompar II. The following settings were used: dendrogram type: unweighted pair group method with arithmetic mean (UPGMA); similarity coefficient: band based: dice; position tolerances: optimization: 4%, position tolerance: 1%. of crops in short rotations. Here, biocontrol agents should solve these problems and help to suppress soil-borne pathogens on a natural way. Although BCAs are already on the market, our biocontrol product will be optimized for desert farming – regarding soil, weather, pathogen species, etc. For this reason, autochthonous bacteria were isolated from rhizosphere and endorhiza of medical plants as well as from bulk soil collected in SEKEM farms, and were evaluated for their potential for biocontrol. In a first step, the dual-culture assay was used to find out the antagonistic potential towards the pathogenic fungi (Berg et al., 2002, 2005a). A total of 1589 bacterial isolates were screened for their ability to inhibit in vitro the growth of Verticillium dahliae, Rhizoctonia solani and Fusarium culmorum. Bacterial isolates obtained from the soil of the SEKEM farm exhibited a higher in vitro antagonistic potential towards soil-borne phytopathogenic fungi in comparison to the bacteria isolated from the desert soil (SEKEM 21.6 ± 0.8%; desert 12.4 ± 0.7%). From the agricultural soil 17.4% (27 isolates) demonstrated antagonism towards all three pathogens, from the desert soil 10.6% (21 isolates) were able to suppress the growth of all fungi tested. Already the desert soil harbours a high proportion of antagonists, which were augmented by organic agriculture in SEKEM soil. The soil from the farm seems to be supplied with antagonists in such an optimal way, that there was no detectable enrichment of antagonists in the rhizosphere and endorhiza of the investigated medical plants. In general, Matricaria


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chamomilla and Solanum distichum showed a better antagonistic potential than Calendula officinalis. Especially the endorhiza from Matricaria chamomilla harbours a high proportion of antagonists. Whereas in the soil and in the rhizosphere could be found most antagonistic bacteria towards Fusarium culmorum, in the endorhiza of the medical plants most antagonists were found towards Verticillium dahliae. In a next step, the antagonistic mechanisms of all isolates, which showed an activity towards at least two of the investigated pathogenic fungi (162 isolates), were investigated in vitro with a special focus on fungal cell wall degrading enzymes (β-1,3-glucanase, chitinase and protease) (Chernin et al., 1995; Grube et al., 2009) and siderophore-production (Schwyn & Neilands, 1987). Production of chitinase could be detected for 8.0% of the antagonists; Lysobacter enzymogenes followed by all isolates of Streptomyces showed a high chitinolytic activity. Glucanase activity was shown for nearly all isolated antagonists (93.8%); only the isolates of the Bacillus cereus group were not able to degrade β-1,3-glucan. Casein degradation by protease could be shown at 80.9% (Bacillus sp. and Lysobacter sp.). The production of siderophores was shown for all antagonists except the isolates of Paenibacillus sp. (93.2%). To avoid investigations with genetically similar strains, amplified rRNA gene restriction analysis (ARDRA) of the 16S rRNA gene with the restriction endonuclease HhaI (Zachow et al., 2008) and BOX polymerase chain reaction fingerprints (Berg et al., 2002; Rademaker & de Bruijn, 1997) of the antagonistic isolates were performed. A representative selection of promising biological control agents was identified by partial 16S rRNA gene sequencing. The use of ARDRA of the 16S rRNA gene with the restriction enzyme HhaI led to the separation of isolates clustered into five groups (data not shown); within groups the similarity of the band patterns was 100% identical: Bacillus subtilis group, Bacillus cereus group, Paenibacillus, Streptomyces and Lysobacter. Except Lysobacter (only one isolate from the rhizosphere of Matricaria chamomilla) only gram-positive antagonists were found. All microenvironments were dominated by antagonists from the Firmicutes branch. Bacillus and Paenibacillus could be isolated from all habitats. Antagonistic isolates of the genus Streptomyces were found exclusively in desert soil. Especially within the large ARDRA cluster of the Bacillus subtilis group containing 123 isolates, analysis of the BOX PCR fingerprints showed a high genotypic diversity. At a cutoff level of 80%, they could be divided into 39 genotypic groups. The genus Paenibacillus could be divided into 11 BOX clusters, Streptomyces was subdivided in three genotypes. According to the ARDRA and BOX dendrograms, 46 preferably genotypically different strains were selected to test them on their antibacterial activity towards Ralstonia solanacearum (Adesina et al., 2007) and Escherichia coli. The cluster of the Bacillus cereus group was completely excluded for further investigations, because of some human pathogenic strains belonging to this taxonomic group. Most isolates of the genus Paenibacillus (identified as P. brasilensis and P. polymyxa) were able to inhibit in vitro the growth of E. coli (7 of 11 isolates), but these strains showed no antagonistic activity towards R. solanacearum. The growth of R. solanacearum was inhibited by 32.6% of the selected antagonists: most isolates of Streptomyces (3 of 4 isolates) and some strains of the Bacillus subtilis group (12 of 30 isolates). Organic amendments like manure, compost and cover crops positively affected the disease suppressiveness of SEKEM soil. During decomposition of organic matter in soil, the ecosystem is subjected to oligotrophication. The ratio of oligotrophic to copiotrophic organisms changes during microbial succession, and this has been associated with general disease suppression (van Bruggen & Semenov, 2000; Garbeva et al., 2004). Our cultivation-

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independent approaches showed an extraordinary high Firmicutes level in SEKEM soils. By cultivation and characterization, the antagonistic role of Bacillus and Paenibacillus (both Firmicutes) was identified. Both are well-known and potent in biocontrol (Berg, 2009; Schisler et al., 2004; Tupinambá et al., 2008). These gram-positive bacteria have a natural formulation advantage due to their ability to form durable, heat-resistant endospores (Emmert & Handelsman, 1999). Lysobacter was the only gram-negative genus identified (Park et al., 2008). This is in contrast to the majority of other studies, where members of the Pseudomonas genus play a major role (Haas & Défago, 2005; Weller et al. 2007). Due to the fact that the proportion of antagonistic strains in soil and root is already high, biocontrol strategies could aim to enhance the diversity of the antagonistic community by application of Lysobacter, Pseudomonas or Serratia strains. However, in our study we selected promising candidates, which will be tested ad planta in comparison to these often used antagonists. 4.2 Strategy to control Fusarium wilt in bananas in Uganda The banana family Musaceae includes monocotyledonous plants of the genera Ensete, Musa and Musella. Most important is the genus Musa comprising 50 to 100 species and cultivars including those with edible fruits like dessert or cooking banana, species with inedible fruits like ornamental bananas or those used for fibres production (Li et al., 2010). In many countries in Africa, Latin America, Asia or the Caribbean, banana production is an important source of income. Banana is the fourth important staple food after rice, wheat and milk in Uganda, the country with the highest per capita consumption per year of cooking banana and the second largest producer after India in the world. Farmers have to deal with several problems as plant pests and diseases, climate change or soil depletion. Diseases caused by fungi, bacteria and viruses are the most limiting factors of high quality production. Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense (Foc), is the most severe disease in banana plants, which leads to high yield losses (Ploetz, 2006). An infestation with the phytopathogen compromises the water and nutrient transport that can cause, in the worst case, the death of the plant. Foc belongs to the F. oxysporum species complex, which is distributed in a broad range of soils and causes serious symptoms on numerous host plants. Despite its ubiquitous occurrence, a morphological identification is difficult and is based primarily on the structure and abundance of asexual reproductive structures and on cultural characterizations (Fourie et al., 2011). The species is divided into more than 150 formae specialis and further subdivided in races, depending on the affected plant cultivars. F. oxysporum persists in soil as immobile chlamydospore until germinating by utilizing nutrients released from plant roots. The life cycle of the fungus commences with a penetration of the spore germ tube or the mycelium of the plants root tip. Further, wounds facilitate the endophyte an entrance of the potential host. When the mycelium entered the xylem vessel, it travels upwards through the plant. In later stages, microconidia are produced, which are distributed in the vessel system and germinate when their movement is stopped. This decreases water and nutrient transport, resulting in severe wilt and eventually death of the plant. Early symptoms of an infestation are reddish brown colouration of the xylem, a yellowing of old leaves and a beginning of wilt. In advanced stages, pseudostem coating leaves collapse and die. The pseudostem sometimes splits. Internally, xylem vessels of the roots and the rhizome turn reddish-brown as the fungus grows through the tissue (Aboul-Soud et al., 2004; Daly & Walduck, 2006). Different studies with bananas and banana plants in vitro and in vivo have shown that plants harbour fungal and bacterial organisms with antagonistic potential towards plant pathogens (Cao et al.,


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2005; de Costa et al., 1997; Lian et al., 2008). However, an efficient strategy to control fungal pathogens especially Foc is still missing. In our study, we used molecular techniques to study banana-associated microbial communities in detail and focus on endophytes, which have a great potential for biocontrol of vascular diseases. For screening of antagonists the rhizosphere, the endosphere and bulk soil of Ugandan banana plants were analysed. The term endosphere refers to the pseudostem of the plant, which is not lignified. Bananas grown in four different fields (variants) in Central Uganda characterized by different manure systems and/or agro-forest systems were sampled. In the first step, bacterial and fungal abundances in the microhabitats were examined. Surprisingly, the highest bacterial abundances with log10 9.4  0.1 g-1 fw were calculated for the endosphere followed by the rhizosphere with log10 8.4  0.3 g-1 fw and soil with log10 7.7  0.3 g-1 fw from R2A medium. Similar values for all microhabitats ranging from log10 6.2  0.2 g-1 fw for rhizosphere followed by soil and endosphere with almost same abundances of log10 5.5  0.3 g-1 fw and log10 5.4  0.3 g-1 fw were estimated for fungal isolates on synthetic nutrient-poor agar (SNA). A total of 1152 bacterial isolates from different media as R2A, MacConkey (for enrichment of Enterobacteriaceae) and King’s B medium (for enrichment of Pseudomonas) and 586 fungi from SNA medium were randomly selected and screened in vitro for their antagonistic potential towards the pathogens. The target pathogen was also isolated from bananas in Uganda. Interestingly, different fungal species were identified: F. oxysporum f.sp. cubense, Fusarium chlamydosporum, and Colletotrichum musae. The latter are known as “low” pathogens; however, strains of all three species were integrated in the screening strategy. The antagonistic activity of bacteria or fungi towards the pathogen evaluated by the method of Berg et al. (2006) ranged from 3 – 6%. Altogether 37 highly active bacterial and 36 fungal strains were further characterized. ARDRA genotyping was able to distinguish bacteria on genus level into Pseudomonas, Bacillus, Burkholderia and Serratia. With repetitive BOX PCR a further characterization on population level was performed. Members of the genus Burkholderia were more diverse than those of Serratia (Fig. 4).

Fig. 4. BOX analysis on species level of bacterial antagonists. First seven isolates were identified as Burkholderia species and the other seven as Serratia marcescens. For identification of isolates the following abbreviations were used: a) habitat with R for rhizosphere, S for soil and E for endosphere, b) number of variant from 1 to 4, c) medium isolated from MC for MacConkey agar, KB for King’s B agar, R2A for R2A agar and SNA for synthetic nutrientpoor agar d) number of replicate from 1 to 4 and e) number of isolate from 1 to 14.

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Additionally, the best antagonists were screened for their ability to produce lytic enzymes like glucanase or protease, which are known for their positive influence in combating fungal pathogens by enzymatic degradation of the cell wall (Kamensky et al., 2003). Further, the production of siderophores, short-chained quorum sensing molecules and the auxin indole-3acetic-acid (IAA) was investigated, which are involved in plant growth promoting processes. The results indicated that 100% of the tested isolates produced an active protease, while only a single isolate, which was identified as Bacillus indicus, was able to degrade glucan. Nearly all strains (94.6%) produced siderophores but only 21.6% isolates, belonging to the genera Pseudomonas and Burkholderia, released quorum sensing molecules. Seven isolates were positively tested for production of IAA, all of them identified as Serratia marcescens. To characterize fungal isolates, morphological groups were identified. Sequencing analysis of the ITS region indicated, that the majority of isolates belong to the genera Penicillium, Paecilomyces, Fusarium and Mortierella. All of them include known biocontrol strains, some actually tested in Musa spp. like non-pathogenic F. oxysporum strains (Kidane, 2008). Cultivation independent analyses include the fingerprint method SSCP, quantitative PCR (qPCR), metagenome analysis and confocal laser scanning microscopy (CLSM) in combination with fluorescence in situ hybridization (FISH). Using SSCP fingerprints, a high specificity was shown for each microenvironment of banana, particularly for the endosphere. The patterns obtained from the bacterial community using universal primers were highly diverse, especially for rhizosphere and soil. This is a typical picture for environmental samples, especially for soil. A detection of bacterial species ranges up to 100 most dominant ones. This problem can be solved by using of more specific primers, e.g. for Pseudomonas or Enterobacteriaceae. Using both in analyses, specific patterns for each habitat appeared. Surprisingly, comparing all fields with different treatments or environmental influence, bacterial, enteric and fungal community didn’t show distinct patterns. This could be explained by a high specificity of banana-associated bacteria independent from the site. The Pseudomonas community was more sensitive, but each site showed an individual pattern. In our study, we found that Enterobacteriaceae were extraordinarily present in and around cultivated banana plants. Therefore, further investigations on the microhabitatspecific communities were performed using a metagenomic approach. The sequences (1944 – 23800) obtained after pyrosequencing were aligned with databases and identified on genus level. In Figure 5 taxa including more than 1% of the totally analysed community were presented. Each habitat harboured a specific arrangement of genera. In the two rhizosphere variants, more than 40% of the identified genera are members of the Enterobacter community, followed by Serratia, Pantoea and Klebsiella with almost 40% and some other genera making up less than 20%. The bacterial composition in the endosphere differed from the rhizosphere samples with a lower number of Enterobacter and higher presence of the genus Raoultella. The highest species richness was shown for the soil sample, with the dominant genus Pantoea with known plant growth promoting species (Bonaterra et al., 2005; Braun-Kiewnick et al., 2000). Serratia, Klebsiella and Enterobacter represented together more than 40% of the analysed species. The analysis illustrates that depending on the investigated microhabitat, different species dominated. For the majority of the listed genera, species with growth promoting abilities are described. In different parts of the plant, diverse species play a key role, like Enterobacter in rhizosphere or Pantoea in soil and endosphere. To complement pyrosequencing data, a further assessment of Pseudomonas and Enterobacteriaceae was performed with quantitative PCR. Similar results were measured for both communities; the highest copy numbers g-1 fresh material of enterics and pseudomonads were detected in endosphere with log10 8.4  0.5 for Pseudomonas and log10 7.9  0.2 for Enterobacteriaceae


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followed by rhizosphere with log10 7.2  0.6 and log10 6.8  0.4 and last with log10 6.3  0.8 for enterics in soil. In the Pseudomonas-specific analysis, no data for soil were received due to values under the detection limit. With confocal laser scanning analysis (CLSM) detection of different bacterial classes as Alpha-, Beta- and Gammaproteobacteria was performed to illustration our data. Due to the fact, that the number of enterics was extraordinary high in prior analyses, the focus in microscopy was also set on Enterobacteriaceae. The microscopic analysis confirmed the previous results, with the detection of a high number of Enterobacteriaceae in the endosphere and also lower detection in rhizosphere.

Fig. 5. Genera of the Enterobacteriaceae community associated with banana plants. Two rhizosphere samples under influence of agro-forest (shaded = S) and not (non-shaded = NS) and one sample from soil and endosphere in comparison. DNA was amplified with entericsspecific primers and analysed by pyrosequencing and identification with the web server SnoWMAn 1.7. The pipeline used was BLAT, NCBI database was selected and included taxa covering more than 1%. Phylogenetic groups accounting for ≤ 1 % of all quality sequences are summarized in the artificial group others. This multiphasic approach showed that the pseudostem of banana – the endosphere – is a unique microenvironment in plants. It is characterized by extremely high microbial abundances, a high diversity and specificity, but a low proportion of antagonistic strains. Enterics play a key role in the bacterial community; they are dominant and represent a cluster of antagonists. However, they also contain human and plant pathogenic species. The endosphere should be the target habitat for biocontrol strategies: the number of strains with a beneficial plant impact should be enhanced here. We have isolated promising strains of Pseudomonas, Bacillus, Burkholderia and Serratia, which are interesting candidates for ad planta experiments. However, it is necessary to pay attention to the enteric community in bananas, especially to the pathogens. 4.3 Strategy to control a multi-species disease in the Styrian oilseed pumpkin Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) is a pumpkin variety that bears its name according to its origin of cultivation that is the Austrian district Styria. The specialty of this cultivar is the absence of a wooden seed shell that facilitates the production of pumpkin seed oil. Beside the culinary aspect of this dark green oil it is

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famous as a very healthy nutritional supplement containing high levels of polyunsaturated fatty acids, antioxidants, vitamins A, B1, B2, B6, C, D, E and counteracts diseases of bladder and prostate. In recent years, dramatic yield losses of Styrian oil pumpkin were reported in Styria due to black rot of pumpkins caused by Didymella bryoniae (Auersw.) Rehm, anamorph Phoma cucurbitacearum (Fr.) (Huss et al., 2007). The ascomycete has a broad host range within the Cucurbitaceae and causes symptoms on vegetative plant parts known as gummy stem blight (Keinath et al., 1995). It spreads from temperate to tropical regions of the world (Sitterly & Keinath, 1996). Fruits, leaves and flower scars are invaded by the pathogen and it can also be seed-borne (Lee et al., 1984; Ling et al., 2010; de Neergaard, 1989; Sitterly & Keinath, 1996). By cultivation-independent SSCP fingerprinting of the fungal ribosomal internal transcribed spacer (ITS) region in combination with DNA sequencing and BLAST analysis (Altschul et al., 1997), it was detected as well in roots of oil pumpkin (data not shown). This underlines the potential establishment of the pathogen even in soils (Bruton, 1998). The analysis of the phenotypic and genotypic variability of the pathogen across different oil pumpkin fields in Styria resulted in a remarkable high morphological versatility in contrast to a low genetic diversity (Zitzenbacher, pers. communication). Styrian oil pumpkins are also affected by bacterial pathogens Pectobacterium carotovorum subsp. carotovorum and subsp. atrosepticum, Pseudomonas spp. and Xanthomonas cucurbitae causing soft rot of pumpkins and leaf diseases (Huss, 2011). The transport of these bacterial phytopathogens by the fungus was observed in vitro (Zitzenbacher, pers. communication) suggesting synergistic interactions between them in the course of co-infections. In order to manage microbial diseases of Styrian oil pumpkin based on autochthonous bacterial and fungal antagonists, initial studies to discover the microbial diversity associated with this host plant were conducted. Roots, female flowers and fruit pulp from three different oil pumpkin cultivars (“Gleisdorfer Ölkürbis”, “Gleisdorfer Diamant” and “GL Maximal”) at a field site in Styria were collected. Root samples were taken at three time points (before flowering, time of flowering, fruits well developed). Bacterial genera Pseudomonas and Bacillus that are known for their plant beneficial interactions (Haas & Défago, 2005) were analysed by SSCP analysis. Data revealed a greater impact of the microhabitat on community structure for Pseudomonas, whereas the plant stage had a stronger impact for Bacillus populations. Female flowers as possible gates for bacterial and fungal infections were analysed in more detail. For Bacillus and Pseudomonas and ascomycete communities, no effect of the plant cultivar on population structure was observed. However, in the flower, the communities are well-structured. FISH-CLSM studies revealed a dense bacterial colonisation of pollen grains that act as propagation vehicles between pistils especially for Alphaproteobacteria (Fig. 6) and shaped in this way the bacterial community structure of the oil pumpkin anthosphere. To obtain oil pumpkin-associated microorganisms for testing their antagonistic properties against D. bryoniae and bacterial pathogens, bacterial and fungal strains were isolated from oil pumpkin cultivars and microhabitats as described above. Endophytes were cultivated from roots and fruit pulp. In addition, seed borne microbial strains were obtained from aforementioned varieties by the isolation from roots, stems and leaves from plants that seeds were surface sterilized and grown under gnotobiotic conditions. Finally 2320 isolates (1748 bacteria and 572 fungi) were subjected to dual culture assays against D. bryoniae A220-2b to test their antagonistic potential against this pathogen. Of tested bacteria, 7.3% inhibited growth, whereas 12.4% of observed fungi showed either growth inhibition or overgrowth of D. bryoniae (Fig. 7).


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Fig. 6. FISH-stained bacteria colonising pollen grains located on pistils of oil pumpkin (GL Opal) visualized by CLSM. A) Alphaproteobacteria (in yellow) and not taxonomically classified bacteria (in red) labelled with ALF968-Cy5 and EUB338MIX-Cy3. B) Alphaproteobacteria labelled with ALF968-Cy5 (yellow), Firmicutes labelled with LGC354MIX-FITC (pink) and taxonomically undefined bacteria (in red) labelled with EUB338Mix-Cy3. C,D) 3D rendered image (Imaris software) of overall bacterial communities (in red) labelled with EUB338MIX-Cy3 and Alphaproteobacteria (red and green) labelled with ALF968-Cy5.

Fig. 7. Amount of oil pumpkin-associated bacterial and fungal isolates positively or negatively tested for in vitro antagonism against D. bryoniae A-220-2b.

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Potential antagonists (128 bacteria and 71 fungi) were subsequently screened in vitro for effects on growth inhibition of Pectobacterium carotovorum subsp. atrosepticum 25-2, Pseudomonas viridiflava 2d1 and Xanthomonas cucurbitae 6h4 to find broad-spectrum antagonists. Altogether, 32% of fungal as well as 49% of bacterial D. bryoniae antagonists were positively tested against at least one, 34% of tested prokaryotes against at least two and 6% of investigated bacterial strains against all three bacterial phytopathogens, whereas no fungal D. bryoniae antagonist was effective against more than one bacterial pathogen (Fig. 8).

Fig. 8. Percentage of fungal and bacterial D. bryoniae antagonists positively tested against at least one, two or all three bacterial pathogens Pectobacterium carotovorum subsp. atrosepticum 25-2, Pseudomonas viridiflava 2d1 and Xanthomonas cucurbitae 6h4. Broad-spectrum antagonists that have the potential to suppress D. bryoniae as well as at least two bacterial phytopathogens of oil pumpkin were characterized genotypically by ARDRA. This resulted in a grouping of 43 bacterial isolates into four different genera: Pseudomonas, Paenibacillus, Serratia and Lysobacter. As a relative high number of isolates belong to Paenibacillus and Lysobacter they were further analysed by BOX PCR (Rademaker & de Bruijn, 1997) to get insight into the intra-genera diversities. Within the group of Paenibacillus a negligible variability between BOX patterns was observed in contrast to strains of Lysobacter that were divided into five groups. Finally five potential broad-spectrum antagonists were chosen for further analysis: one representative for Pseudomonas, Paenibacillus and Serratia and two representatives from the Lysobacter cluster. Partial sequencing of 16S rRNA genes with subsequent BLAST analysis (Altschul et al., 1997) was performed for their identification and the following species could be affiliated to respective strains: Pseudomonas chlororaphis P34, Paenibacillus polymyxa PB71, Serratia plymuthica S13, Lysobacter antibioticus L175 and L. gummosus L101. To learn more about the mode of antagonism of chosen broad-spectrum antagonists against D. bryoniae, dual culture assays in which growth inhibition of D. bryoniae A-220-2b by either soluble or volatile antimicrobial compounds secreted by the five test strains was assessed were performed. Results suggest a high capability of broad-spectrum antagonists to synthesize bioactive compounds: sterile culture supernatants from P. chlororaphis P34, L. gummosus L101 and P. polymyxa PB71 as well as volatile organic compounds (VOCs) excreted from these bacteria and S. plymuthica S13 as well suppressed growth of the fungus significantly compared to control treatments (ANOVA; LSD, p < 0.05; data not shown).


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Performances of broad-spectrum antagonists in terms of promoting plant growth and health will facilitate the selection of bacterial strains that will be analysed for the production of a biological strengthener for Styrian oil pumpkin. Studies with the model organism C. elegans (Zachow et al., 2009) will give insight into the potential pathogenicity of remaining test strains. The manufacture of the final product will further depend on the finding of an appropriate formulation procedure that guarantees a high stability of the ultimate BCAs/PGPRs.

5. Conclusion Advanced ecological knowledge about plant-associated microorganisms and interactions of the biocontrol agent(s) with abiotic and biotic factors support the development of efficient biocontrol strategies. As shown in three examples, specific strategies have to be developed adapted to the life cycle of the pathogen and the autochthonous microbial communities in the target habitat. The latter varied strongly dependent on the plant species, microenvironment and climate.

6. Acknowledgement The project regarding biocontrol in the desert was funded by the EU-Egypt Innovation Fund. We would like to thank our co-workers of the Libra Company and the SEKEM farms for good cooperation, and the founder of SEKEM – Ibrahim Abouleish – for inspiring discussions. The banana project in Uganda was funded by the Federal Ministry of Finance (BMF) of the Republic of Austria through the Austrian Development Agency (ADA). Here, we thank our colleagues in Uganda: Sam Mpiira and John Baptist Tumuhairwe (Kampala) for help with the sampling. The Styrian pumpkin project was funded by the Austrian State (Lebensministerium) and the regional government of Styria. We want to thank Johanna Winkler (Gleisdorf) for providing us oilseed pumpkin seeds, Eveline Adam, Sabine Zitzenbacher (Graz) for assistance with field trials and pathogens, Athanassios Mavridis (Göttingen) and Herbert Huss (Raumberg-Gumpenstein) for providing pathogens. From our institute we would like to thank Massimilliano Cardinale, Christin Zachow and Henry Müller for their relevant support.

7. References Aboul-Soud, M.A., Yun, B.W., Harrier, L.A. & Loake G.J. (2004). Transformation of Fusarium oxysporum by particle bombardment and characterisation of the resulting transformants expressing a GFP transgene. Mycopathologia 158, 475-482 Adesina, M.F., Lembke, A., Costa, R., Speksnijder, A. & Smalla K. (2007). Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: site-dependent composition and diversity revealed. Soil Biol Biochem 39, 2818-2828 Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J.H., Zhang, Z., Miller, W. & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402 Amann, R.I., Ludwig, W. & Schleifer, K.H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59, 143-169

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2 Development of RNAi in Insects and RNAi-Based Pest Control 1Institute

Guang Yang1, Minsheng You1, Liette Vasseur1,2, Yiying Zhao3 and Chunhui Liu1

of Applied Ecology, Fujian Agriculture and Forestry University 2Department of Biological Sciences, Brock University 3College of Agriculture, Shihezi University 1,3China 2Canada

1. Introduction In agricultural systems, insect pests can cause crop damage mainly through loss in yield or quality resulting in a loss in profits for farmers. Worldwide pests cost billions of dollars due to damage and use of pesticides. Chemical pesticides are still the major approach for controlling insect pests, but they are associated with significant hazards to the environment and human health. The alternative commercial biotechnological system relies mostly on the expression of Bacillus thuringiensis insecticidal proteins (Cry toxins). Its effectiveness however is threatened by the development of resistance in some species such as Ostrinia nubilalis (Lepidoptera, Pyralidae) and Heliothis virescens (Lepidoptera: Noctuidae) (Ferre and Van Rie, 2002; Baum et al., 2007). As a result, there is an urgent need to develop economically and ecologically sound alternatives for pest control. Gene silencing has been suggested as one of the new alternatives to reduce damage from insect pests. RNA interference (RNAi) is first described by Fire et al. (1998), and its mechanism lies in that a double-stranded RNA (dsRNA) introduced in an organism has the capacity to silence post-transcriptional genes (Hannon, 2002; Geley and Muller, 2004). RNAi is highly conserved in eukaryotic organisms (Fire, 2007). It is considered as a specific type of defence mechanism (Terenius et al., 2011). Four different types of RNAi have been described including short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), endogenous siRNAs (endo-siRNAs or esiRNAs), and microRNAs (miRNAs) (Terenius et al., 2011). To date, RNAi has been proven promising for research on gene function determination and gene knockdown in eukaryotes and medical control of cancers and viral disease (Huvenne and Smagghe, 2010). In insects, studies have mainly targeted the understanding of the RNAi mechanism, and the function, regulation and expression of genes. Introduction of dsRNA into an organism has been tested by using different techniques such as microinjection (Bettencourt et al., 2002;

1

These authors contributed equally to this chapter.

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Tomoyasu and Denell, 2004; Ghanima et al, 2007), soaking, or, most preferably, oral feeding of artificial diet (Eaton et al., 2002; Turner et al., 2006; Baum et al., 2007; Mao et al., 2007; Chen et al., 2008; Tian et al., 2009). Transgenic plants producing dsRNAs directed against genes function in Lepidoptera, Coleoptera, and Hemiptera pests are becoming more common (Gordon and Waterhouse, 2007; Baum et al., 2007; Mao et al., 2007, Chen et al., 2010). For example, Chen et al. (2010) report the successful feeding of TPS (trehalose-6-phosphate synthase for the synthesis of trehalose, main sugar reserve in haemolymph) dsRNA solutions to silence this gene thus proposing it as a useful pest control agent. These results suggest that over time, new generations of insect-resistant crops will be created to manage agriculturally important insect pests. In this review chapter, we summarize the current knowledge on the recent RNAi research on insects, including the application of RNAi techniques in research involving functional insect genes and functional genomics, the methods of dsRNA uptake RNAi in insects, the systemic diffusion of RNAi silencing molecules in the insect body and the mechanism underlying this diffusion, and the potential application of RNAi in integrated pest management (IPM). The main purpose of this review is to help entomologists become familiar with RNAi research, a rapidly growing field where new avenues and techniques are being used to investigate insect RNAi mechanisms for the development of pest control.

2. Study on the function of insect genes using RNAi methods RNAi is a powerful tool for the study on the function of insect genes. It was first used in the study of a model insect, the fruit fly Drosophila melanogaster (Lipardi et al., 2001). RNAi studies in D. melanogaster have laid a solid foundation for the development of insect RNAi technology and the elucidation of the RNAi mechanisms in insects. Recently, Huvenne and Smagghe (2010) have reviewed the definitions of RNAi in insects while Terenius et al. (2011) have analyzed the variability and the implications of over 150 published and unpublished studies, mainly focusing the analysis on lepidopteran insects, on the need for further studies on RNAi mechanisms. In this section, we discuss a selected group of published studies and the main orientations used by researchers in exploring these techniques. Table 1 summarizes the studied functions, methods used for RNAi introduction and the main responses of insects. Besides D. melanogaster, 20 other insect species are reported here, including 7 species of Lepidoptera, 3 species of Coleoptera, 3 species of Orthoptera, 2 species of Hymenoptera, 2 species of Homoptera, 1 species of Diptera, 1 species of Isoptera, and 1 species of Hemyptera. These selected papers have all in common the successful use of RNAi mechanisms as potential pest control agent. It is important to note that, except for a few exceptions (e.g. circadian clock gene), most studies have targeted different genes. Responses also greatly vary from minor effects such as disruption in functional rhythm to reduction in fitness and increased mortality. As reported by Terinius et al. (2011) for lepidopteran species, most of the studies have helped better understand developmental processes and the immune system.

3. Internal diffusion of RNAi molecules within insect body The effects of RNAi inside the body of insects are determined by an important factor, the spread of silencing RNA molecules inside the insect body (so-called systemic RNAi). In

29

Development of RNAi in Insects and RNAi-Based Pest Control

Insects Silkworm Bombyx mori

Genes and References Circadian clock gene per (Sandrelli et al., 2007) Ecdysis-triggering hormone gene ETH (Dai et al., 2008) β-actin gene (Gvakharia et al., 2003) Circadian clock gene per (Kotwica et al., 2009) Carboxylesterase gene EposCXE1 and pheromone binding protein gene EposPBP1 (Turner et al., 2006) Cytochrome P450 gene CYP6AE14 (Mao et al., 2007) Glutathione-S-transferase gene GST1 (Mao et al., 2007)

Methods

Effects Disruption of egg-hatching Transgenics rhythm Transgenics

Lethal at pharate second-instar larval stage

Injection

Disruption of sperm release

Injection

Delayed sperm release

Feeding

Inhibition of gene expression

Feeding

Inhibition of larval growth

Feeding

Successful inhibition of gene expression

Beet armyworm Chitin synthase gene Spodoptera exigua (Chen et al., 2008)

Injection

Disorder in the insect cuticle, no expansion of the larval trachea epithelial wall, and other larval abnormalities

Japanese pine sawyer Monochamus alternatus

Injection

Pupal and adult cuticle sclerotisation, death at a high dose

Egyptian cotton leafworm Spodoptera littoralis Light brown apple moth Epiphyas postvittana Cotton bollworm Helicoverpa armigera

Red flour beetle Tribolium castaneum

Laccase gene MaLac2 (Niu et al., 2008)

Chitin synthase genes TcCHS1 and TcCHS2 (Arakane et al., 2005)

Injection

Chitinase-like proteins TcCHT5, TcCHT10, TcCHT7, and TcIDGF4 (Zhu et al., 2008)

Injection

Disruption in all types of moulting(larva-larva, larvapupa, and pupa-adult), cessation of ingestion, decrease in larval size, and reduction of chitin content in the midgut Effects on pupal-adult moulting Effects on egg hatching, larval moulting, pupation, and adult metamorphosis. Effects on abdominal contraction and wing/elytra extension. Effects on adult eclosion

30 Insects Western corn rootworm Diabrotica virgifera virgifera LeConte Striped flea beetle Phyllotreta striolata Mediterranean field cricket Gryllus bimaculatus

German cockroach Blattella germanica American grasshopper Schistocerca americana Brown planthopper Nilaparvata lugens Turnip sawfly Athalia rosae


Genes and References

Methods

Effects

Vacuolar ATPase (v-ATP) Feeding (Baum et al., 2007)

Delayed larval development and increased mortality

Arginine kinase gene AK (Zhao et al., 2008)

Feeding

Delayed development, increased mortality, and reduced fertility

Injection

Complete loss of circadian control of locomotor activity and electrical activity in the optic lobe

Circadian clock gene per (Moriyama et al., 2008)

Nitric oxide synthase Injection gene NOS (Takahashi et al., 2009) BgRXR gene (Martin et Injection al., 2006) Pigment-dispersing factor Injection gene pdf (Lee et al., 2009)

Destruction of long-term memory

Eye colour gene vermilion (Dong and Friedrich, Injection 2005)

Suppression of ommochrome formation and systematic expression

Trehalose phosphate synthase (TPS) (NlTPS Feeding mRNA) (Chen et al., 2010) Ar white gene (Sumitani et Injection al., 2005)

European honey Transcription factor gene Relish (Schlüns and bee Apis mellifera Crozier, 2007) Salivary nitrophorin 2 Triatomid bug gene NP2 Rhodnius prolixus (Araujo et al., 2006) Savannah tsetse TsetseEP gene and fly transferrin gene 2A192 Glossina morsitans (Walshe et al., 2009) morsitans

Injection

Inhibition of pupal eclosion Effects on insect night activity

Disturbed development through disruption in the TPS enzymatic activity, reduction of insect survival rate White phenocopy in embryonic eye pigmentation Inhibition of Relish gene expression and reduction in the expression of two other immune genes, abaecin and hymenoptaecin

Injection and feeding

Shortened plasma coagulation time

Feeding

Inhibition of TsetseEP gene expression, but no inhibition of 2A192 gene expression

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Insects Eastern subterranean termite Reticulitermes flavipes

Genes and References Methods Cellulase enzyme gene Cell-1 and casteregulatory hexamerin Feeding storage protein gene Hex2 (Zhou et al., 2008)

Effects Reduction in group fitness and increased mortality

Table 1. RNAi research on functional genes in insects plants, the nematode Caenorhabditis elegans, and the planarian Schmidtea mediterranea, RNAi is systemic as the RNAi signal spreads throughout the entire biological system by travelling between cells (Fire et al., 1998; Newmark et al., 2003). In insects, RNAi is not always to be systemic. For example, fly cells take up dsRNA, which cannot spread throughout the entire body (Saleh et al., 2006). Whangbo and Hunter (2008) have defined different mechanisms for dsRNA uptake: cell-autonomous and non-cell autonomous. Huvenne and Smagghe (2010) described these two types of RNAi and their level of spread which would be greater in systemic non-cell autonomous RNAi than in the cell autonomous RNAi. While most research in insects has been conducted with cell-autonomous RNAi, it is suggested that studies should focus towards non-cell autonomous RNAi as a better potential for defining agent of insect control. In summary, studies have shown that the ability to distribute an RNAi signal is different in different insects. The intake of dsRNA by Drosophila cells leads to localised gene silencing, without systemic distribution of the RNAi signal (Van Roessel et al., 2002; Roignant et al., 2003; Dietzl et al., 2007). On the other end, Tribolium (Tomoyasu et al., 2008) and Schistocerca americana (Dong and Friedrich, 2005) have strong systemic RNAi reactions. The gene responsible for nematode systemic RNAi is sid-1 (Winston et al., 2002). Correspondingly, the sid-1 gene is not found in the Drosophila genome, whereas the grasshopper has a sid-1 ortholog (Dong and Friedrich, 2005), and Tribolium also has a sid-1-like gene (Tomoyasu et al., 2008). Further BLAST searches at the NCBI website have identified one species in Coleoptera, one in Lepidoptera, two in Hymenoptera, and three in Hemiptera containing sid-1 homologs, whereas no homologous gene has been found in Homoptera (Walshe et al., 2009). Further studies have found that the RNAi mechanisms in Tribolium and the nematode C. elegans are different. Tribolium does not have some of the key elements that are required for RNAi in C. elegans, such as RNA-dependent RNA polymerase (RdRP) and the RNA channel transporter (SID) (Fire et al., 1998; Winston et al., 2002). Furthermore, the function of the sid-1like gene of Tribolium is not to absorb RNAi but, instead, is similar to the function of the tag130 gene of C. elegans (Tomoyasu et al., 2008). Therefore, further verification is needed to define the function of the sid-1 gene in insect RNAi. Recent studies have shown that the anti-viral RNAi reaction in Drosophila depends on a virus-specific immune signal and systemic spreading (Saleh et al., 2009). Further studies need to be conducted to understand the spread of silencing RNA within the insect body and the genes involved in this process. Understanding and revealing the molecular mechanisms of determining how RNA spreads systemically inside the insect body will facilitate the application of RNAi technology for pest control.

4. Methodology of dsRNA uptake in insects Methods of dsRNA uptake in insects can greatly vary and strongly influence the efficiency of gene silencing, thus their potential as insect pest control agent. It is important to note that

32


since gene silencing is only limited to cells that are infected, the main challenge is the selection of the delivery system (Terenius et al., 2011). In both types, methods of delivery must be first defined, being effectively easier and better understood for cell-autonomous RNAi machinery (Siomi and Siomi, 2009). The main uptake (or delivery) methods include injection, soaking, feeding, transgenic technique, and viral infection. This section examines these various mechanisms and their effectiveness in delivering RNAi and gene silencing in various species. 4.1 Microinjection Microinjection, i.e. the direct injection of dsRNA into the body of insects, has been one of the most effective delivery methods for systemic RNAi types. Short dsRNA have had the most success with this mechanism (Siomi and Siomi, 2009). In addition, the 5’ end of the dsRNA can affect the effectiveness of RNAi; a phosphorylated 5’ end exhibits better gene silencing rate than does a hydroxylated 5’ end (Boutla et al., 2001). The major advantage of injecting dsRNA into the insect body is the high efficiency of inhibiting gene expression. There are however some limitations with micro-injection. First, the cost for in vitro synthesis and storage of dsRNA is relatively high, and the steps are complicated. In addition, injection pressure and the wound generated inevitably affect the insects. It has been shown that skin damage stimulates the immune response. In practice, this delivery method would have very limited application as pest control agent. 4.2 Soaking Soaking D. melanogaster embryos in a dsRNA solution can inhibit gene expression, and its effectiveness is comparable to the injection method in that it requires a higher concentration of dsRNA (Eaton et al., 2002). Soaking D. melanogaster S2 cells in CycE and ago dsRNA solutions has been shown to effectively inhibit the expression of these two genes for cell cycle, thereby elevating levels of protein synthesis (March and Bentley, 2007). The soaking method is suitable only for certain insect cells and tissues as well as for specific insects of developmental stages that readily absorb dsRNA from the solution, and therefore, it is rarely used. 4.3 Feeding of artificial diet Compared to other methods, dsRNA feeding is the most attractive primarily because it is convenient and easy to manipulate. Since it is a more natural method of introducing dsRNA into insect body, it causes less damage to the insect than microinjection (Chen et al., 2010). It is especially popular in very small insects that are more difficult to manipulate using microinjection. Early insect RNAi feeding studies were frustrating; for example, the injection of dsRNA effectively silenced the aminopeptidase gene slapn, which is expressed in the midgut of Spodoptera littoralis, but feeding with dsRNA did not achieve RNAi (Rajagopal et al., 2002). Fortunately, there are other studies showing that dsRNA feeding can be successful for RNAi studies in insects. Feeding dsRNA to E. postvittana larvae has been shown to inhibit the expression of the carboxylesterase gene EposCXE1 in the larval midgut and also to inhibit the expression of the pheromone-binding protein EposPBP1 in adult antennae (Turner et al., 2006). dsRNA feeding also inhibite the expression of the nitrophorin 2 (NP2) gene in the salivary gland of Rhodnius prolixus, leading to a shortened coagulation time of plasma


33

(Araujo et al., 2006). dsRNA feeding has also been successful in many other insects, including insects of the orders Hemiptera, Coleoptera, and Lepidoptera (Baum et al., 2007; Mao et al., 2007). The main challenge remains that there needs to be a greater amount of material for delivery as silencing has been shown to be incomplete (Chen et al., 2010). This phenomenon has been observed after ingestion of CELL-1 dsRNA by the termite Reticulitermes flavipes (Zhou et al., 2008), TPS dsRNA in N. lugens nymphae (Chen et al. 2010), Nitrophorin 2 dsRNA by Rhodnius prolixus (Araujoa et al., 2006). In addition, different species of insects have different sensitivities to RNAi molecules when delivered orally. For example, Glossina morsitans fed with dsRNA may effectively inhibit the expression of TsetseEP in the midgut, but cannot inhibit the expression of the transferrin gene 2A192 in fat bodies due to lack of transfer capacity between tissues (Walshe et al., 2009). The mechanisms associated with the transfer of gene expression through feeding delivery method still need further study. In addition, one method that may be better than direct feeding with dsRNA is the use of transgenic plants to produce dsRNA (Baum et al., 2007; Mao et al., 2007). The advantage of this method is the generation of continuous and stable dsRNA material. Genetically engineered dsRNA-producing yeast strains have also been developed to feed D. melanogaster, but gene silencing was not successful (Gura, 2000). However, dsRNA produced in bacteria is effective in C. elegans (Timmons and Fire, 1998). Therefore, the use of bacteria, especially insecticidal microorganisms, to produce dsRNA for insect RNAi merits further study. 4.4 Developing transgenic insects The advantage of using transgenic insects that carry the dsRNA is that as it is inheritable, the expression can be stable and continuous. The technique has been proposed to help either reduce population through introduction of sterile insects or for population replacement. In this case, dsRNA must be first injected in the host insect. Tests are being conducted on several species with promising results but as stated by Scolari et al. (2011), there is a need to understand environmental and genetic influences when assessing the potential use of such transgenics. The transgenic method has been first used in D. melanogaster with the GAL4/UAS transgenic system that leads to the expression of hairpin RNA (Kennerdell and Carthew, 2000; Tavernarakis et al., 2000). Subsequently, transgenic technology has generated transgenic Aedes aegypti that produces dsRNA (Travanty et al., 2004). Through the use of a U6 promoter in D. Melanogaster, S2 cells can generate short hairpin RNA (shRNA) to inhibit gene expression (Wakiyama et al., 2005). RNAi molecules targeting the circadian clock gene per have also introduced into Bombyx mori embryos by a piggyback plasmid to obtain genesilenced transgenic individuals (Sandrelli et al., 2007). The transfection technique has been used to silence the D. melanogaster mitochondrial frataxin gene dfh, generating large-sized, long-lived larvae and short-lived adults (Sandrelli et al., 2007). The GAL4/UAS transgenic system has also been used in B. mori (Sandrelli et al., 2007) to allow for induction of the transgenic construct. Therefore, gene function can be studied within a certain time period, and the study of gene functions in development, physiology, and the nervous system is possible. 4.5 Virus-mediated uptake Virus-mediated RNAi methods involve the infection of the host with viruses carrying dsRNA formed during viral replication and targeting the gene of interest in the host. For

34


example, recombinant Sindbis virus introduced into B. mori cells through electroporation can produce dsRNA to inhibit BR-C gene expression, causing the larvae not to pupate or leading to adult defects (Uhlirova et al., 2003). Virus-mediated RNAi studies are still rare. However, this method takes advantage of the infection and ability of the virus to spread rapidly in a host population. Virus-mediated RNAi does not require screening for transgenic insects or tissues, and thus, it has unique advantages.

5. RNAi-based pest control In our struggle to minimizing the damage caused by insect pests, we have to acknowledge that pests cannot be efficiently managed by utilizing a single pest control agent. Several studies have shown that pest resistance to chemical pesticide and more recently to Bt has increased requiring new techniques to be applied to reduce the impacts of pest on crop production. While commonalities regarding the development of resistance to chemical and biological control agents remain to be determined, research suggests that both biochemical and genetic factors can contribute to this resistance. It is therefore crucial to continue examining the potential of integrated pest control or management (IPM) to reduce the threat of pests on agroecosystems. IPM has been suggested as a strategy to control incidence of pests since the 1950’s and is based on six components: controlled pest populations, healthy crops, monitoring, mechanical and biological controls, and responsible use of pesticides (Kogan, 1998). The basis of for IPM is to balance ecological gain with economic loss (Southwood and Way, 1970). Over the past decade, the number of studies examining these issues has been increasing with, RNAi, as a novel pesticide-free way, to be integrated into IPM. Research is still in its infancy to examine the application of RNAi machinery for pest control, as most of the focus has been on functional studies of insect genes. Huvenne and Smagghe (2010) however describe in some detail the potential application of RNAi in insect control through cell-line and feeding-in-plant experiments. Based on the currently available literature, they suggested five important factors largely influencing the silencing effect and the efficiency of RNAi as insect pest control technique: concentration of dsRNA, nucleotide sequence, length of dsRNA fragment, persistence of the silencing effect, and life stage of the target pest. Through insect gene function studies involving injecting or feeding with dsRNA, we have found that some gene silencing can dramatically affect insect growth and development. Theoretically it would be possible to use RNAi to inhibit insect gene leading to insect control. Already, pest control using transgenic plants expressing dsRNA have been published (Baum et al., 2007; Mao et al., 2007). Transgenic corn expressing dsRNA against the vacuolar ATPase gene (v-ATP) significantly decreases the damage caused by D. virgifera virgifera LeConte and, notably, protects corn crops (Baum et al., 2007). Introduction of RNAi elements targeting the CYP6AE14 gene, which is directly related to gossypol detoxification in Helicoverpa armigera, into Arabidopsis or tobacco inhibits CYP6AE14 gene expression in H. armigera feeding on the transgenic plants and, therefore, increases the toxicity of gossypol (Mao et al., 2007). Although there needs to be more testing in the field and at large scale, transgenic insects have also been tested as a mechanism for pest control (Scolari et al., 2011). For both transgenic plants and insects, very few species have been investigated and it is clear that further research is essential to explore the potential use of transgenics as an effective means for pest control. Like any other control mechanisms, risk assessment will be


35

required to determine whether RNAi technology as a form of pest control will be safe and likely create a new era in pest control.

6. Conclusion Widespread increase in the application of RNAi technology in insect research has facilitated the identification of insect gene function. Research has shown that while dsRNA is particularly conservative, there are various functions and development factors among insect species. Such variations are yet to be fully understood but certainly can serve as a basis for determining their capacity to control insect genes. The main challenge for moving towards larger scale projects remains the development of effective delivery mechanisms. Feeding is very popular in insect RNAi research and may have the most promising future in pest control, especially with the creation of transgenic plants producing dsRNA. Overtime, the use of transgenic insects will also lead to more efficient pest control. Our understanding of the types of dsRNA and their spreading mechanisms within an organism can limit our ability to move further. Indeed, existing studies have not provided enough evidence that systemic RNAi, with silencing RNA molecules spreading throughout the entire body, can be achieved in all insects. Which insects have characteristics promoting systemic RNAi? Are the mechanisms underlying systemic RNAi the same in different insect species? Such questions need to be answered before moving further in developing large scale pest control systems. Undoubtedly, there is broad potential for the application of RNAi technology in pest control, mainly if combined into IPM strategies.

7. Acknowledgements This work was supported by the National Key Project of Fundamental Scientific Research in China (“973” Programs, No. 2011CB100404), and the projects of the National Natural Science Foundation of China (No. 30871649, 30970528, and 30971925).

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3 Evaluation of Plant Extracts on Mortality and Tunneling Activities of Subterranean Termites in Pakistan Sohail Ahmed1, Mazhar Iqbal Zafar1, Abid Hussain3, Muhammad Asam Riaz1 and Muhammad Shahid2

1Department

2Department

of Agri. Entomology, University of Agriculture, Faisalabad, of Chemistry and Biochemistry, University of Agriculture, Faisalabad, 3College of Natural Resources and Environment, South China Agricultural University, Guangzhou, 1,2Pakistan, 3P. R. China

1. Introduction Plant extracts offer a vast, virtually untapped reservoir of chemical compounds with many potential uses. One of these uses is in agriculture to manage pests with less risk than with synthetic compounds that are toxicologically and environmentally undesirable. Increasing evolution of resistance in pest population further derives the need to search for new bioactive compounds with a wide range of new modes of action. Various experiments using plant extracts in human and animal health protection, agriculture and household pest management have been particularly promising (Pascual-Villalobos & Robledo, 1999; Scott et al., 2004). The apparent societal hope for using plant extracts in place of more traditional pesticides has also increased the attention paid to natural products in the past decade (Duke et al., 2003). Plant products have been exploited as insecticides, insect-repellents, antifeedants and insect growth and development regulators (Saxena, 1998). The deleterious effects of phytochemcials or crude plant extracts on insects are manifested in several ways, including suppression of calling behaviour (Khan & Saxena, 1986), growth retardation (Breuer & Schmidt, 1995), toxicity (Hiremath et al., 1997), oviposition deterrence (Zhao et al., 1998), feeding inhibition (Wheeler & Isman, 2001) and reduction of fecundity and fertility (Muthukrishnan & Pushpalatha, 2001). Many plants have been recognized to have anti-termitic activities (Sakasegawa et al., 2003, Park & Shin, 2005, Jembere et al., 2005, Cheng et al., 2007, Ding & Hu, 2010, Supriadi and Ismanto, 2010) or repellent to the termites i.e., Eucalyptus globules, lemmon grass, Eucalyptus citrodora, cedar wood, clove bud and vetiver grass (Zhu et al., 2001a, b), Taiwania cryptomerioides Hayat (Chang et al., 2001), Dodonaea viscosa (Purple hop bush) a termite resistant shrub (Anonymous, 2001), Ocimum basilicum L., Cymbopogon winterianus Jowitt, Cinammomum camphora, Rosmarinus officinalis (Sbeghen et al., 2002) and Coleus ambionicus (Singh et al., 2004) are less extensively studied against termites.

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The extracts of plants having anti-termite properties and termite-resistant formulations have been prepared, reported and tested in the laboratory and fields. Substrates in these tests were soil, sand and filter paper. Mortality and inhibition of consumption of wood were indicators of toxic and feeding deterrent activity of these extracts. Those tested in laboratory were extracted in various organic solvents in addition to water. Alcoholic and phenolic compounds in extracts of Juniperus procera (Kinyanjui et al., 2000), pine resin and eight of its derivatives (Nunes et al., 2004), 2.0 % chloroform leaf extracts of Polygonum hydropiper L. and Pogostemon parvillorus against tea termite, Odontotermes assamensis Holm. with highest toxic activity (100% mortality) in the extract of P. hydropiper (Rahman et al., 2005) are some of the examples. Effects of hexane, ethanol, and petroleum ether extracts of the black pepper fruits, Piper nigrum, were studied on the dry-wood termite, Cryptotermes brevis. Hexane extract at 0.5% concentration induced 50% mortality, which dropped to 4.76 and 14.28% with ethanol and petroleum ether, respectively, 2 days post treatment (Moein & Farrag, 2000). The termite, Coptotermes curvignathus, workers responded differently to soils and pine blocks treated with varying concentrations of Azadirachta excelsa leaf extracts in acetone, hexane and methanol. The result showed that extracts from A. excelsa leaves had an inhibitory effect on subterranean termites, C. curvignathus. The soils treated with the extracts did pose a hindrance to the tunneling activities of the termites (Sajap and Aloysius, 2000). The chloroform extracts of the woods Ipe (Tabebuia sp.) and Itauba (Mezilaurus sp.) on the drywood termite, Cryptotermes brevis, applied at a rate of 0.1 g/mL to filter paper to feed the termites and later analyses of substrate consumption rates and mortality by the KruskalWallis method indicated a statistically significant reduction of feeding rates and increased mortality after 30 days (Cabrera et al., 2001). Phytoextracts from Adhatoda vasica, Cynodon dactylon, Pongamia pinnata, Rauvolfia serpentina, Cleistanthus collinus, Tamarindus indica and Eichhornia crassipes controlled the termites, Microcerotermes mycophagus (Nilanjana & Chattopadhyay, 2003). The biological activity of extracts of Meliaceae in relation with Heterotermes tenuis was studied in the laboratory. The effect of aqueous extracts from Melia azedarach, Trichilia pallida and Azadirachta indica (neem) (1 and 5% w/v), neem oil (1 and 2% v/v), and Nimkol, obtained from neem leaves (0.5 and 1% a.i.), were measured for the survivability of the termites. Nimkol caused a significant mortality of H. tenuis after the third day of feeding (1% a.i.) (Castiglioni and Vendramim, 2003). Seed and leaf extract of Polygonum hydropiper and Cannbis sativa against Heterotermes indicola and Coptotermes heimi showed more toxicity of seed (52-64% and 70-74% mortality) than leaf extracts (28-54 % and 28-58%) in both species. Crude extracts of various reproductive and vegetative parts of Calotropis procera had toxic effects on H. indicola (Badshah et al., 2004). Datura alba, D. stramonium and Calotropis procera were the most effective against the termites with 62.5% protection (Bajwa & Rajpar, 2001; Ayodele & Oke, 2003). 5% Chloroform extract of Lantana camara var. aculeate at a concentration of 5% was found to be significantly effective against termite workers (Verma & Verma, 2006). The extracts used in the field were mostly in the form of decoction with water and were used in the soil or poured into the termites’ nest directly. Decoction of Cassia fistula, Myrtus communis, Sapium sebiferum and Thevetia peruviana at rate of 5% (5g: 100ml) provided significant protection against termites for three months in the field. Fermented extracts of Tithonia diversifolia and Melia azedarach controlled Isopteran insects when poured into their nest. The ash of T. diversifolia, Cassia siamea and C. spectabilis applied to affected trees provided protection from termites for up to 45 days. Vernonia amygdalina and Agave sisalana, not only controlled termites and but also contributed to soil fertility (Ghosh, 2009). Soil treated with 2% solution of

Evaluation of Plant Extracts on Mortality and Tunneling Activities of Subterranean Termites in Pakistan

41

Calotropis procera L. and Azadirachta indica prevented damage to sugarcane setts by Odontotermes obesus (Rambur) controlled the termite (Deka & Singh, 2001; Singh et al., 2002). Several novel classes of termiticides have been isolated from plants and based on these natural products, more active analogs have been synthesized. Two sesquiterpenes (partheniol and argentone) and a triterpene (incanilin) from guayule resin showed different levels of antifeedant and toxic activity (Gutierrez et al., 1999). The effects of a commercial insecticide formulation (margosan-O) containing 0.3%. Azadirachtin and 14% neem oil on orientation, tunneling, and feeding behaviour of the Formosan subterranean termite have been investigated (Grace & Yates, 1992). Sand treated with vetiver oil or nootkatone at 100 g/g substrate were effective barriers to the termite, Coptotermes formosanus (Maistrello et al., 2001). Thiophenes from four Echinops species and columellarin and sesquiterpene lactone fraction from the heartwood of Australian white cypress (Callitris glaucophylla) showed anti-termitic activities against C. formosanus Shiraki (Watanabe et al., 2005; Fokialakis et al., 2006)). Vulgarone B (isolated from Artemisia douglasiana ), apiol (isolated from Ligusticum hultenii) and cnicin (isolated from Centaurea maculosa) exhibited significantly higher mortalities in Formosan subterranean termite (C. formosanus) than in untreated control in the laboratory bioassay (Meepagala et al., 2006). Oils extracted from plant parts have been applied in a number of situations to protect the substrate from termite infestation. The crude seed oil of Piper guineense, each at a 10% concentration at the rate of 18 litres ha-1 significantly lowered damage by termites (Microtermes spp., Macrotermes bellicosus and M. subhyalinus) (Umeh & Ivbijaro, 1999). Neem seed oil inhibited growth of termite surface-tunnels (Yashroy & Gupta, 2000). For further references, annual meeting report of IRG can be consulted for efficacy of oils against termites. Many timbers contain chemicals or complex mixture of chemicals that repel or kill the termites or effect on gut flora in termites (Adams et al., 1988); among these are Pometia pinnata, Homalium foetidum, Eucalyptus deglupta and Alstonia scholaris (Rokova and Konabe., 1990). Relatively less mentioned other plants with termite control properties are presented below (Anonymous, 2001). Species Carya ovata Cedrela odorata Consolida regalis Dodonaea viscosa Quercus prinus Hardwickia mannii Pinus strobus Samadera indica Carica papaya Grevillea robusta Leucaena leucocephala Commiphora Africana Cassia siamea Hyptis spicigera Ocimum canus Source: HRD Publication UK

Parts Used Bark Wood wood Leaves, wood / pulp Bark Stem/ branches Bark Leaves Fruit, fresh leaves and roots Leaves Used as a leaf mulch Gum/ resin Used as a leaf mulch Aerial parts Whole plant

Property Termiticidal Termiticidal Termiticidal Termiticidal Termiticidal Termiticidal Termiticidal Termiticidal Insecticidal Insecticidal Repellent Repellent Repellent Repellent Insecticidal, repellent

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Many plant extracts have been found to alter the behaviour of termites. Chemicals showing antifeedant activities had also effect on tunneling of the termites (Ibrahim et al., 2004; Mao & Henderson, 2007). The foregoing examples are just crust of the copious literature available on this aspect of termite management and control. Previously we have demonstrated some of the above mentioned properties of many extracts of plants, shrubs and trees in our laboratory and have found significant results in controlling termites in the field (Ahmed et al., 2005, 2006, 2007). In order to find out inexpensive alternate to synthetic insecticides, anti termite properties from plants will continue to expand base of the effective molecules to be developed to go well with the ecology of termites.

2. Materials and methods 2.1 Collection of termites The assorted workers of the termite species, Microtermes obesi Holm., in the later instars were collected, within the damaged canes from sugarcane fields and from the corrugated cardboard baits in PVC monitors installed in the fields at different places at the Experimental Area, Department of Agri-Entomology, University of Agriculture, Faisalabad. 2.2 Following plants were selected to obtain their leaf extracts Botanical name Adhatoda vasica (Nees) Dodonaea viscosa (Linn.) Jacq Thevitia peruviana (Pers) Merr Nerium odorum Soland Salvadora oleiodes Decne Alstonia scholaris (R. BR.) Delphinium ajacis Linn. Papaver somniferum Linn. Lucaena leucocephala (Lam.) DeWit Grevilla robusta A.Cunn.Ex.R.Br Tephrosia purpurea Linn. Nerium oleander Linn. Jatropha integerrima Jacq.

Family Acanthaceae Sapindaceae Apocynaceae Apocynaceae Salvadoraceae Apocynaceae Ranunculaceae Papaveraceae Mimosaceae. Proteaceae Fabaceae Apocynaceae Euphorbiaceae

Common name Malabar nut Hopbush Yellow oleander Indian oleander Vann Devil tree Larkspur Garden poppy Iple iple Silky oak Wild indigo Rose bay Peregrina

2.3 Extraction method 2.3.1 Preparation of leaves for extraction process Fresh fully developed leaves in the season from middle portion of the plants from Botanical Garden as well as from areas within campus, University of Agriculture, Faisalabad, Pakistan, were collected and these plants were never exposed to pesticides. These leaves were washed with tap water and then air dried in a laboratory for 2 weeks ensuring sufficient air flow to avoid damping. The room-dried leaves were reduced to a powder form by grinding with an electric grinder running at a speed of 6000 rpm for 5060 sec.


43

2.3.2 Crude methanolic extract of leaves One hundred gram (100 g) of powder from each of the plants was extracted in 200 ml of 80% methanol in the ratio of 1:2 (w/v) by following method of extraction (Sadek, 2003). It was kept for 72 hours at room temperature and shaken at intervals to get a better extraction. Thereafter, the extract was filtered through Whatman filter No. 42. After filtering, the methanol was removed at 60°C using rotary evaporator, to obtain solid extract, dried in vacuum desiccator. The final yield of dry material was used to prepare percent solution of crude extract with 2% methanol. 2.3.3 Aqueous extracts of leaves To get the aqueous extracts, above procedure was followed except powder was extracted in distilled water. The filtrates were stored in a refrigerator at 5°C for subsequent use in bioassays. 2.4 Bioassay 2.4.1 Soil preparation for bioassay The soil used in bioassays was sandy clay loam (52.6% sand, 24.8% silt and 20.6% clay). There had been no known applications of agro-chemicals in this soil for the control of termites. The soil was sieved through a 30-mesh screen and moisture was determined with the help of a moisture meter. Water was added in this soil to simulate 50% of water holding capacity, to avoid mortality of termites due to dehydration during assays. 2.4.2 Bioassays by mixing leaf extract in the soil Antitermitic sugarcane strip bioassays (ASSB) using different leaf extracts were done in Petri dishes (95 × 15 mm) containing 20 g sifted sterilized soil and strips of sugarcane (1.5 cm × 6 cm) to keep the termites alive. Every treatment with 10%, 20% and 40% of extracts and control (without extract) were repeated thrice in Completely Randomized Design (CRD). 20 g of sifted soil in Petri dish having sugarcane strip was wetted/ mixed with respective concentration of the extract. 50 active workers and 5 soldiers were released in the Petri dishes having treated and untreated soil. 2.4.3 Filter paper bioassay Whatman filter paper No. 42, 9 cm in diameter was treated with 10, 20 and 40% concentrations of leaf extracts at the rate of 31 µl/cm2 and placed in Petri dishes (95 × 15 mm). 50 workers and 5 soldiers of were released in the Petri dishes having treated and untreated filter paper. The Petri dishes having filter paper and/or soil bioassay were placed in growth chamber under controlled conditions of 28±2°C and 80%±5 humidity. Data for mortality were recorded after every 2 hours up to 12 hours, and then after every 12 hours until all workers and soldiers died. Each treatment was repeated three times. 2.5 Formation of Galleries (FG) Members of family Termitidae make galleries during foraging. This shows the activity of termites in the soil. The termites started making tunnel along the bottom of each Petri dish around the sugarcane strip. Termite’s response towards galleries formation for each plant extract at each concentration after 5, 10 and 15 hours was determined by plotting the tunnels


44

on the cellophane paper and measured the length in mm2 with the help of planimeter. The values were correlated with the chemical concentrations. Tunnelling activities were analyzed by Factorial Analysis (CRD). 2.6 Statistical analysis LT50s in soil and filter paper bioassay was determined using Kaplan Meier Survival Test. In all tests, values of LT50s among replication was non significant and were thus taken as mean LT50. Tunnelling activities analyzed by Two way ANOVA, however, data are represented as mean activity of all time intervals at each concentration. The difference among concentrations was determined by Duncan Multiple Range Test (DMR) at p80% % of LT50 on filter paper and in the soil when compared with control treatments. In other studies, leaf extracts of Jatropha integerrima, N. oleander and Lucaena leucocephala in acetone, methanol, petroleum ether and aqueous solvents showed activity in terms of mortality of termite workers at different concentrations when mixed in the soil in Petri dishes and is represented in Tables 5-7. J. integerrima was the most effective among three plants and showed lowest LT50 at 10% (9.72 hours) in acetone and then in petroleum ether at 20 and 40% concentrations (6.53 and 4.99 hours, respectively). Lowest LT50 of two other plants was shown in acetone extract at 40% concentration (47.8 hours). It is interesting to note that N. oleander has shown less activity than N. odorum, in addition to acetone and petroleum ether, in methanol and aqueous extracts as well.


46

T. purpurea G. robusta

a

P. somniferum

LT50 (hrs)

300 200 100 0 0%

10%

20%

40%

Plants x concentrations

T. purpurea G. robusta

b

P. somniferum

LT50 (hrs)

300 200 100 0 0% 10% 20% 40% Plants x concentrations

Fig. 1. LT50 values with methanolic extract of Tephrosia purpurea, Papaver somniferum, Grevilla robusta leaves at different concentrations against M. obesi on (a) filter paper (b) mixed in the soil. T. purpurea G. robusta

a

P. somniferum

LT50 (hrs)

300 200 100 0 0% 10% 20% 40% Plants x concentrations

LT50 (hrs)

b

T. purpurea P. somniferum G. robusta 300 200 100 0 0% 10% 20% 40% Plants x cocentrations

Fig. 2. LT50 values with aqueous extract of Tephrosia purpurea, Papaver somniferum, Grevilla robusta leaves at different concentrations against M. obesi in Petri dishes having treated (a) filter paper (b) soil.


Concentrations

47

LT50 (hours)

(%)

acetone

methanol

aqueous

0 10 20 40

396.9 13.8 8.6 6.7

483.5 9.7 5.4 4.5

457.4 79.6 17.9 15.9

petroleum ether 483.5 108.6 6.5 4.9

Table 5. LT50 values of various extract of Jatropha integerrima at different concentrations against M. obesi in Petri dishes having treated soil. Concentrations

LT50 (hours)

(%)

acetone

methanol

aqueous

0 10 20 40

154.0 123.1 85.8 85.6

174.8 165.2 130.0 116.2

162.2 128.2 127.0 94.9

petroleum ether 177.5 143.2 142.0 138.7

Table 6. LT50 values of various extract of Nerium oleander at different concentrations against M. obesi in Petri dishes having treated soil. Concentrations (%) 0 10 20 40

acetone 149.0 84.5 64.6 47.8

LT50 (hours) aqueous 182.0 133.7 125.7 99.0

methanol 167.4 152.0 146.0 135.7

petroleum ether 176.9 147.8 147.5 133.0

0% 400

a

10% a

a

a

20%

40% a

a

a

a

a

300 200 100

b

b

cd

b c d

c

b d

c

b

d

b c

c

d

b d

b

c d

cd

b

c d

.r ob us ta G

eu co ce ph al P. a so m ni fe ru m

ch ol ar is

L. l

A. s

le io de s

S. o

D .a ja ci s

pe ru vi an a

T.

.o do r N

A. v

um

0

as ic a

Tunnel length (mm 2)

Table 7. LT50 values of various extract of Leucaena leucocephala at different concentrations against M. obesi in Petri dishes having treated soil.


Fig. 3. Comparison of tunnel length at different concentrations of leaf methanol extracts of various plants.


0% 400

a

10% a

a

a

20%

40%

a

a

a

a

a

300 200 100

bc

bc

d

b

d

bc

cd

b d

c

b

c

d

bc

d

bc

d

b d

c

d

.r ob us ta

eu co ce ph al P. a so m ni fe ru m

G

L. l

A. s

S. o

ch ol ar is

le io de s

.a ja ci s D

pe ru vi an a

T.

N .o do r

A. v

um

0 as ic a


48


Fig. 4. Comparison of tunnel length at different concentrations of leaf aqueous extracts of various plants. a


0% 600

a

400

ab bc

10% a

20% a

ab bc

c

c

ab bc

40% a c

b c c

200 0 Acetone

Methanol Aqueous Petroleum ether Solvents x concentrations


b

0% 600 400

a a b

b

10%

20%

a a

a ab

b

b

40% a c c

b

c

c

200 0 Acetone

Methanol

Aqueous Petroleum ether

Solvents x concentrations

Fig. 5. Comparison of termites’ tunnel length in leaf extracts in various solvents of (a) Nerium oleander (b) Lucaena leucocephala. Various concentrations had significant difference among them with respect to the tunnelling (tunnel length) by the termites when mixed with the soil at various time intervals (5-15


49

minutes). After 15 minutes, it was difficult to draw the tunnel length on the paper, however, data are shown as mean tunnel length of the time intervals but not of the concentrations. A. vasica, N. odorum, S. oleiodes, T. purpurea and G. robusta leaf extracts in methanol at 40% did not show any tunnelling, nevertheless, termites had mined in the aqueous extracts of the same plants at 40% concentration (Figs. 3 & 4). In contrast to N. odorum, other species of the same plant N. oleander could not prevent tunnel formation as in case of former species, but tunnel length at 40% concentrations had significant difference than that at 10 and 20% concentrations depending upon type of solvent (Fig. 5 a & b).

4. Discussion Treatment of soil with natural/synthetic compounds to control the termites is common method. Insecticides have been used to form barrier in soil against subterranean termites to prevent their tunnelling and reaching to food sources. Chlorpyrifos, bifenthrin, fipronil and many others have been extensively used for this type of barrier against termites (Su et al., 1997). Criteria used to evaluate potential soil termiticides have been termites’ ability to tunnel through treated soil and toxicity of material (plant extracts) in laboratory experiments (Grace et al., 1993; Su et al., 1993). The results exhibited herein are mostly the confirmation of the results obtained elsewhere for the medicinal plant extracts having anti-termite properties and termite-resistant formulations (Singh et al., 2001; Ding & Hu, 2010). Dodonaea viscosa (Purple hop bush) has been reported as a termite resistant shrub (Anonymous, 2001), but bioassay of its extracts with termites has been investigated for the first time in this report. The extracts from Adhatoda vasica and Nerium oleander are some of the above mentioned plants which have been tested for same purpose (Nilanjana & Chattopadhyay, 2003). The results revealed two important aspect of toxicological inference (i) LT50 irrespective of medium for feeding and movement was almost equal and non-significant depending upon fiducial limits (ii) LT50 of methanol extract was shorter than aqueous extract. Many studies have shown activity of plant extracts when applied on filter paper and / or mixed in soil to determine mortality (Blaske & Hertel, 2001; Blaske et al., 2003, Jembere et al., 2005) and concluded that plant extracts have the potential for under- and above-ground application for the termite control. The present results showed that the tunnelling activities are the function of time and concentration. All concentrations of aqueous and methanolic leaf extracts have less tunnelling activities of M. obesi as compared to control. Means of the tunnelling activities of M. obesi in methanolic extract treated soil were less than the means of the tunnelling activities in aqueous extract treated soil. There was no tunnelling in leaf methanol extracts of A. vasica, N. odorum, S. oleiodes, T. purpurea and G. robusta at their 40% concentrations. These results are confirmation of earlier reports mentioined elsewhere depending upon species and kind of plant parts being studied. It is evident from these results that extracts did pose a hindrance to tunnelling activities of the termites (Sajap & Aloysius, 2000; Maistrello et al., 2001; Peterson & Ems-Wilson, 2003; Mao & Henderson, 2007), but the termites, however, may become insensitive towards the extracts upon longer period of exposure and this period depends upon termites species. The extracts usually oils have been reported for toxicity and tunnelling inhibition studies, however, extracts in water and organic solvents have also yielded results for the above properties for termites’ control. It has been summarized from various studies that extracts


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having both properties of being toxic and inhibit tunnelling is good for various types of application and can be used in baiting and media application. There is no denying that potential application against termites would require large volume of plant materials, thus a number of plants should be studied to use alternatively. The water extract may be used for delivery into the soil to directly kill termites, or a paint-on material which may prevent termites from infesting wood. Inhibition of tunnelling may be exploited in a number of situations in agricultural ecosystem where seed or plant parts may be prevented from access of termites. One such example is setts protection from plant extract against termites where significant germination of setts of sugarcane in plots treated with plant extracts than in control plots (Ahmed et al. 2005; 2007).

5. Conclusions 1. 2.

3.

Laboratory bioassays with a range of plant extracts in particular indicated the potential of some of them as termiticides. Plant extracts used in the present studies had repellency for termites as these checked the tunnelling activities of termites in the soil, and may be used to keep the termites away from plants and ultimately, saving the plants from damage. Leaf extracts of D. viscosa D. ajacis and N. oleander can be the good candidates for further process of isolation, and characterization of active compounds in the extracts.

6. References Adams, R.P., McDaniel, C.A. & Carter, F.I. (1988). Termiticidal activities in the heartwood, bark/sapwood and leaves of Juniperus species from the United States. Biochemical Systematics and Ecology, Vol. 16, pp. 453-456, ISSN 0305-1978. Ahmed, S., Naseer, A. & Fiaz, S. (2005). Comparative efficacy of botanicals and insecticides on termites in sugarcane at Faisalabad. Pakistan Entomologist, Vol.27, pp. 23-25, ISSN 1017-1827. Ahmed, S., Khan, R.R. & Riaz, M.A. (2007). Some studies on the field performance of plant extracts against termites (Odontotermes guptai and Microtermes obesi) in sugarcane at Faisalabad. International Journal of Agriculture and Biology, Vol.9, pp. 398–400, ISSN 1560-8530. Ahmed, S., Riaz, M.A. & Shahid, M. (2006). Response of Microtermes obesi (Isoptera: Termitidae) and its gut Bacteria towards some plant extracts. Journal of Food Agriculture & Environment, Vol. 4, No.1, pp. 317-320, ISSN 1459-0255. Anonymous, 2001. Termite conrol without chemicals. HDRA-the organic organisation, pp: 10-12. Ayodele, M.S. & Oke, O.A. (2003). Studies on the potential of some plant-based community pest management strategies in Southwest Nigeria. An investigation of the antitermite potency of Datura stramonium L. An International Journal of Agricultural Science, Science, Environment and Technology, (ASSET) Series B: Vol.2, No.2, pp.153-159, ISSN 1595-9694. Badshah, H., Farmanullah, Salihah, Z., Aur Saljoqi & Shakur, M. (2004). Toxic effects of AK (Calotropis procera) plant extracts against termites (Heterotermes indicola and Coptoternes heimi) (Isoptera: Rhinotermitidae). Pakistan Journal of Biological Science, Vol.7, No.9, pp.1603-1606, ISSN 10288880


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Bajwa, G.A & Rajpar, M.N. (2001). Biological activity of extact of different plants against termites, nettle tree leaf beetle and amaltas leaf stitcher. Pakistan Journal of Forestry, Vol.51, No.2, pp.31-41, ISSN 0030-9818. Blaske, V.U. & Hertel, H. (2001). Repellent and toxic effects of plant extracts on subterranean termites (Isoptera: Rhinotermitidae). Journal of Economic Entomology, Vol.94, No.5, pp.1200–1208, ISSN 0022-0493. Blaske, V.U., Hertel, H. & Forschler, B.T. (2003). Repellent effect of isoborneol on subterranean termites (Isoptera: Rhinotermitidae) in soils of different composition. Journal of Economic Entomology, Vol.96, No.4, pp.1267-1274, ISSN 0022-0493. Breuer, M. & G.H. Schmidt, G.H. (1995). Influence of a short period treatment with Melia azedarach extract on food intake and growth of the larvae of Spodoptera frugiperda (Lepidoptera; Noctuidae). Journal of Plant Diseases and Protection, Vol.102, pp.633654, ISSN 1866-3829. Cabrera, R.R., Lelis, A.T. & Berti-Filho, E. (2001). Effects of extracts of woods Tabebuia sp. (bignoniaceae) and Mezilaurus sp. (Lauraceae) on the dry-wood termite, Cryptotermes brevis (Isoptera: Kalotermitidae). Arquivos-do-Instituto-Biologico-SaoPaulo, Vol.68, No.1, pp.103-106, ISSN 0020-3653 Castiglioni, E. & Vendramim, J.D. (2003). Evaluation of repellence of Heterotermes tenuis (Isoptera: Rhinotermitidae) to Meliaceae by-products. Agrociencia. Montevideo, Vol.7, No.1, pp.52-58, ISSN 1510-0839. Chang, S.T., Cheng, S.S. & Wang, S.Y. (2001). Antitermitic activity of essential oils and components from Taiwania (Taiwania cryptomerioides). Journal of Chemical Ecology, Vol.27, No.4, pp.1267-1274, ISSN 0098-0331. Cheng, S.S., Chang, H.T., Wu, C.L. and Chang, S.T. (2007). Anti-termitic activities of essential oils from coniferous trees against Coptotermes formosanus. Bioresource Technology, Vol.98, pp.456-459, ISSN 0960-8524. Deka, M.K. and Singh, S.N. (2001). Neem formulation in the management of sugarcane insects and pests. Proceeding of 63rd American Convention Sugar Technologist Association, pp. 33-38. 27th – 28th August, Jailpur, India. Ding, W. & Hu, X.P. (2010). Antitermitic effect of the Lantana camara plant on subterranean termites (Isoptera: Rhinotermitidae). Insect Science, Vol.17, No.5, pp. 427–433, ISSN 1744-7917. Duke, S.O., Baerson, S.R., Dayan, F.E., Rimando, A.M., Scheffler, B.E., Tellez, M.R., Wedge, D.E., Schrader, K.K., Akey, D.H., Arthur, F.H., Lucca, A.J., Gibson, D.M., Harrison, H.F., Peterson, J.K., Gealy, D.R., Tworkoski, T., Wilson, C.L. & Morris, J.B. (2003). ARS Research on Natural Products for Pest Management. Pest Management Science, Vol. 59, No.6-7, pp.708-717, ISSN 1526-498X. Fokialakis, N., Osbrink, W.L.A., Mamonov, L.K., Gemejieva, N.G., Mims, A.B., Skaltsounis, A.L., Lax, A.R. & Cantrell, C.L. (2006). Antifeedant and toxicity effects of Thiophenes from four Echinops species against the Formosan subterreanean termite, Coptotermes formosanus. Pest Management Science, Vol.62, No.9, pp.832-838, ISSN 1526-498X. Ghosh, G.K. (2009). Termite control in Kenya. In: Biopesticide and Integrated Pest Management. pp. 199-204, Aph Publishing Corporation. India. ISBN 8176481351.

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Grace, J.K. & Yates, J.R. (1992). Behavioural effects of neem insecticide on Coptotermes formosanus (Isoptera: Rhinotermitidae). Tropical Pest Management, Vol.38, No.2, pp.176-180, ISSN 0143-6147. Grace, J.K., Yates, J.R., Tamashiro, M. & Yamamoto, R.T. (1993). Persistence of organochlorine insecticides for Formosan susterranean termite (Isoptera: Rhinotermitidae) control in Hawaii. Journal of Economic Entomology, Vol.86, No.3, pp. 761-766, ISSN 0022-0493. Gutierrez, C., A. Gonzalez-Coloma, A. & Hoffmann, J.J. (1999). Antifeedant properties of natural products from Parthenium argentatum, P. argentatum X P. tomentosum (Asteraceae) and Castela emoryi (Simaroubeaceae) against Reticulitermes flavipes industrial. Industrial Crops and Products, Vol.10, No.1, pp.35-40, ISSN 0926-6690. Hiremath, I.G., Youngjoon, A., Soonll, K., Ahn, Y.J. & Kim, S.I. (1997). Insecticidal activity of Indian plant extracts against Nilaparvata lugens (Homoptera: Delphacidae). Applied Entomology and Zoology, Vol.32, No.1, pp.159-166, ISSN 0021-4914. Ibrahim, S., Kambham, S., Henderson, G. & Jayasimha, P. (2004). Toxicity and repellency of sesame oil on Formosan subterranean termites (Isoptera: Rhinotermitidae) in treated sand. Proceedings of the National Conference on Urban Entomology, 61-63, Phoenix, AZ. 20-22 May, USA. Jembere, B., Getahun, D., Negash, M. & Sevoum, E. (2005). Toxicity of Birbira (Milletia ferruginea) seed crude extracts to some insect pests as compared to other botanical and synthetic insecticides. 11th NAPRECA (Natural Products and Drug Delivery) Symposium Book of Proceeding, Astanarivo, Madagaskar, pp. 88-96. Khan, Z.R. & Saxena, R.C. (1986). Effect of steam distillate extracts of resistant and susceptible rice cultivars on behaviour of Sogatella furcifera (Homoptera: Delphacidae). Journal of Economic Entomology, Vol.79, No.4, pp.928-935, ISSN 00220493. Kinyanjui, T., Gitu P.M. & Kamau, G.N. (2000). Potential antitermite compounds from Juniperus procera extracts. Chemosphere, Vol.41, No.7, pp.1071-1074, ISSN 0045-6535. Mao, L. & Henderson, G. (2007). Antifeedant activity and acute and residual toxicity of alkaloids from Sophora flavesces (Leguminosae) against Formosan subterranean termites (Isoptera: Rhinotermitidae). Journal of Economic Entomology, Vol.100, No.3, pp. 866-870, ISSN 0022-0493. Maistrello, L., G. Henderson, G. & R.A. Laine, R.A. (2003). Comparative effects of vetiver oil, nootkatone and disodium octaborate tetrahydrate on Coptotermes formosanus and its symbiotic fauna. Pest Management Science, Vol.59, No.1, pp.58-68, ISSN 1526-498X. Maistreollo, L., G. Henderson, G. & R.A. Laine, R.A. (2001). Efficacy of vetiver oil and nootkatone as soil barriers against Formosan subterranean termite (Isoptera: Rhintermitidae). Journal of Economic Entomology, Vol.94, No.6, pp.1532-1537, ISSN 0022-0493. Meepagala, K.M., Osbrink, W., Sturtz, G. & Lax, A. (2006). Plant- derived natural products exhibiting activity against Formosan subterranean termites. Pest Management Science, Vol.62, No.(6), pp.565-570, ISSN 1526-498X. Moein, S.I. & Farrag, R.M. (2000). Susceptibility of the dry-wood termite Cryptotermes brevis Walker to the black pepper extracts. Egyptian Journal of Agricultural Research, Vol.78, No.3, pp.1135-1140, ISSN 1110-6336.


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Muthukrishnan, J. & Pushpalatha, E. (2001). Effects of plant extracts on fecundity and fertility of mosquitoes. Journal of Applied Entomology, Vol.125, pp.31-35, ISSN 09312048. Nilanjana, D. & Chattopadhyay, R.N. (2003). Control of termites through application of some phyto-extracts – a new approach in forestry. Indian Forester, Vol.129, No.12, pp.1538-1540, ISSN 0019-4816. Nunes, L., Nobre, T., Gigante, B. & Silva, A.M. (2004). Toxicity of pine resin derivatives to subterranean termites (Isoptera: Rhinotermitidae). Environment Quality Management, Vol.15, No.5, pp.521-528, ISSN 1520-6438. Park, I.K. & Shin, S.C. (2005). Fumigant activity of plant essential oils and components from garlic (Allium sativum) and clove bud (Eugenia caryophyllata) oils against the Japanese termite (Reticulitermes speratus Kolbe). Journal of Agriculture, Food and Chemistry, Vol.53, pp. 4388-7392, ISSN 0021-8561. Pascual-Villalobos, M. & Robledo, A. (1999). Anti-insect activity of plant extracts from the wild flora in south-eastern Spain. Biochemical Systematics and Ecology, Vol.27, pp.110, ISSN 0305-1978. Peterson, C.J. & Ems-Wilson, J. (2003). Catnip essential oil as a barrier to subterranean termites (Isoptera: Rhinotermitidae) in the laboratory. Journal of Economic Entomology, Vol.96, No.4, pp.1275-1282, ISSN 0022-0493. Rahman, I., Gogoi, I., Dolui, A.K. & Handique, R. (2005). Toxicological study of plant extracts on termite and laboratory animals. Journal of Environmental Biology, Vol. 26, No.2, pp.239-241, ISSN 0254-8704. Rokova, M. & Konabe, C. (1990). Assessment of untreated Papua New Guinea timbers for resistance to subterranean termites. Papua New Guinea Forest Research Institute. Klinkii. Vo.4, No.2. pp.19-27. Sadek, M.M., (2003). Antifeedant and toxic activity of Adhatoda vasica leaf extract against Spodoptera littoralis (Lep. Noctuidae). Journal of Applied Entomology, Vol.127, pp.396404. ISSN 0931-2048. Sajap, A.S. & Aloysius, F. (2000). Effects of leaf extracts of Azadirachta excelsa on Coptotermes curvignathus (Isoptera: Rhinotermitidae). Sociobiology, Vol.36, No.3, pp.497-503, ISSN 0361-6525. Sakasegawa, M., Hori, K. & Yatagi, M. (2003). Composition and anti-termite activities of essential oils and Melaleuca species. Journal of Wood Science, Vol.49, pp. 181-187, ISSN 1435-0211. Saxena, R. C. (1998). Botanical pest control, In: Critical issues in Insect Pest Management,, Dhaliwal G.S. & Heinrichs, E.A. (Eds), 115-179, New Delhi, India. Sbeghen, A.C., Dalfovov, V., Serafini, L.A. & De-Barros, N.M. (2002). Repellence and toxicity of basil, citronella, ho-sho and rosemary oils for the control of the termite, Cryptotermes brevis (Isoptera: Kalotermitidae). Sociobiology, Vol.40, No.3, pp.585594, ISSN 0361-6525. Scott, I.M., Jensen, H., Nicol, L., Bradbury, R., Sanchez-Vindas, P., Poveda, L., Arnason, J.T. & Philogene, B.J.R. (2004). Efficacy of piper (Piperaceae) extracts for control of common home and garden insect pests. Journal of Economic Entomology, Vol.97, No.4, pp. 1390-1403, ISSN 0022-0493.

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Singh, G., Singh, O.P., Prasad, Y.R., de-Lampasona, M.P. & Catalan, C. (2004). Chemical and insecticidal investigations in leaf oil of Coleus amboinicus Lour. Journal of Flavour and Fragrance, Vol.17, No.6, pp.440-442, ISSN 0882-5734. Singh, Y., Ranawat, B.S., Verma, R.K. & Nayal, S.S. (2001). Termite control with medicinal plant products. Journal of Medicinal and Aromatic Plant Sciences, Vol. 22/23, No.4A/1A, pp.151-153, ISSN 6253-7125. Singh, M., Lal, K. Singh, S.B. & Singh, M. (2002). Effect of Calotropis (Calotropis procera) extract on infestation of termite (Odontotermes obesus) in sugarcane hybrid. Indian Journal of Agricultural Sciences, Vol.72, No.7, pp.439-441, ISSN 0019-5022. Su, N.Y., Chew, V., Wheeler, G.S. & Scheffrahn, R.H. (1997). Comparison of tunneling responses into insecticide-treated soil by field populations and laboratory groups of subterranean termites (Isoptera: Rhinotermitidae). Journal of Economic Entomology, Vol.90, No.2, pp.503-509, ISSN 0022-0493. Su, N.Y., Scheffrahn, R.H. & Ban, P.M. (1993). Barrier efficacy of pyrethroid and organophophate formulations against subterranean termites (Isoptera: Rhinotermitiae). Journal of Economic Entomology, Vol.86, No.3, pp.772-776, ISSN 0022-0493. Supriadi and Ismanto, A. (2010). Potential Use of Botanical Termiticide. Perspektif, Vol.9, No.1, pp. 12-20. ISSN: 1412-8004. Umeh, V.C. & Ivbijaro, M.F. (1999). Effect of termite damage to maize of seed extracts of Azadirachta indica and Piper guineense in farmers’ field. Journal of Agricultural Science, Vol.133, No.4, pp.403-407, ISSN 0021-8596. Verma, R.K. & Verma, S.K. (2006). Phytochemical and termiticidal study of Lantana camara var. aculeata leaves. Fitoterapia, Vol.77, No.6, pp.466-468, ISSN 0367-326X. Watanabe, Y., Mitsunaga, T. & Yoshimura, T. (2005). Investigating antitermitic compounds from Australian white cypress heartwood (Callitris glaucophylla) against Coptotermes formosanus Shiraki. Journal of Essential Oil Research, Vol.17, No.3, pp.346-350, ISSN 1041-2905. Wheeler, D. A. & Isman, M. (2001). Antifeedant and toxic activity off Trichilia americana extract against the larvae of Spodoptera litura. Entomologia Experimentalis et Applicata, Vol.98, pp.9-16, ISSN 0013-8703. Yashroy, R.C. & Gupta, P.K. (2000). Neem-seed oil inhibits growth of termite surfacetunnels. Indian Journal of Toxicology, Vol.7, No.1, pp.49-50, ISSN 0971-6580. Zhao, B., Grant, G.G., Langevin, D. & MacDonald, L. (1998). Deterring and inhibiting effects of quinolizidine alkaloids on the spruce budworm (Lepidoptera: Tortricidae) oviposition. Environmental Entomology, Vol.27, pp. 984-992, ISSN 0046-225X. Zhu, B.C.R., Henderson, G., Chen, F., Fei, H. & Laine, R.A. (2001a). Evaluation of vetiver oil and seven insect active essential oils against the Formosan subterranean termite. Journal of Chemical Ecology, Vol. 27, No.8, pp.1617-1625, ISSN 0098-0331. Zhu, B.C.R., Henderson, G., Chen, F., Maistrello, L. & Laine, R.A. (2001b). Nootkatone is a repellent for Formosan subterranean termite (Coptotermes formosanus). Journal of Chemical Ecology, Vol. 27, No.3, pp.523-531, ISSN 0098-0331.

4 Botanical Insecticides and Their Effects on Insect Biochemistry and Immunity Arash Zibaee

Department of Plant Protection, College of Agriculture, University of Guilan, 41635-1314, Rasht Iran 1. Introduction Some concerns, especially environmental ones, lead the researchers to find new avenues of insect control in agriculture. Considering negative effects of synthetic pesticides especially on non-target organisms caused a general perception that natural compounds are better products or Generally Regarded As Safe (GRAS) (Scott et al., 2003). So, researches has been concentrated on the plant kingdom for solutions leading to the production of a myriad of secondary compounds that can have toxic, growth reducing, and antifeedant properties against insects (Berenbaum & Zangerl, 1996). The use of plant extracts (botanical insecticides) to protect crops and stored products is as old as crop protection. Indeed, prior to the development and commercial success of synthetic insecticides beginning in the 1940s, botanical insecticides were major weapons in the farmer’s arsenal against crop pests (Isman, 2008). Four major types of botanical insecticides are being used for insect control including pyrethrum, rotenone, neem, and essential oils along with three others in limited use (Isman, 2006). Pyrethrum is an oleoresin extracted from the dried flowers of the pyrethrum daisy, Tanacetum cinerariaefolium (Asteraceae) that its active ingredients are three esters of chrysanthemic acid and three esters of pyrethric acid (Isman, 2006). The insecticidal action of the pyrethrins is characterized by a rapid knockdown effect, particularly in flying insects, and hyperactivity and convulsions in most insects. These symptoms are the result of the neurotoxic action of the pyrethrins, which block voltage-gated sodium channels in nerve axons. Azadirachtin is an extraction from Indian neem tree, Azadirachta indicahas that has two profound effects on insects (Schmutter, 2002). Azadirachtin, apart from its antifeedant effects on insects, inhibited the synthesis and release of ecdysteroids from the prothoracic gland resulting incomplete ecdysis in immature insects and sterility in adult females (Isman, 2006). Rotenone is a type of isoflavonoids extracted from the roots or rhizomes of the tropical legumes like Derris, Lonchocarpus, and Tephrosia (Isman, 2006). Rotenone is a mitochondrial poison by blocking the electron transport chain leading to inhibition of energy production (Hollingworth et al., 1994). Acetogenin extracts from seeds of Annona squamosa known as annonin I, or squamocin, and a similar compound, asimicin were isolated from the bark of the American pawpaw tree, Asimina triloba (Johnson et al., 2000). Although, there are many plant extracts widely use against insects but here one of them, Artemisia, is discussed. The genus Artemisia is a member of a large plant family Asteracea

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(Compositae) encompassing more than 300 different species of this diverse genus. Several isolated compounds from this species have shown anti-malarial, antibacterial, antiinflammatory, plant growth regulatory and cytotoxicity (antitumor) activities (Akhtar and Isman, 2004).

2. Effect of botanical insecticides on digestive enzymes of insects Digestion is a process in which ingested macromolecules by insects break down to smaller ones to be absorbable via epithelial cells of midgut. Several enzymes based on food materials have critical roles in this process. Any disruption in their activity disables insects to provide their nutrients for biological requirements. Several studies demonstrated the effect of botanical insecticides on feeding parameters of insects by demonstrating food consumption [CR = I/DT], approximate digestibility of consumed food [%AD = 100(I–F)/I], efficiency of converting the ingested food to body substance [%ECI = 100 G/I], efficiency of converting digested food to body substance [%ECD = 100G/(I–F)] and consumption index [CI = I/W] (Shekari et al., 2008). The fact underlying these changes is inhibitory effects of botanical insects on digestive enzymes (Zibaee and Bandani, 2010a). Starch in plants and glycogen in animals are the storage carbohydrates that amylases are necessary to digest them in herbivorous and carnivorous insects, respectively. a-Amylases (EC 3.2.1.1) catalyze the endohydrolysis of long a-1,4-glucan chains such as starch and glycogen (Terra and Ferriera, 2005). Saleem & Shakoori (1987) showed that sublethal concentrations of pyrethroids decreased the a-amylase activity in the larval gut of the beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). Shekari et al. (2008) demonstrated that a-amylase activity level in elm leaf beetle treated by A. annua extract decreased after 24 h and sharply increased after 48 h. Zibaee & Bandani (2010a) showed that Artemisia annua extract caused the reduction of a-amylase activity in Eurygaster integriceps Puton (Hemiptera: Scutelleridae), and this reduction increased by higher concentrations of plant extract. After amylase, glycosidases digest carbohydrate oligomers to monosaccharides (Terra and Ferriera, 2005; Zibaee et al., 2008a; Zibaee et al., 2009a). On the other hands, glycosidases catalyze the hydrolysis of terminal, non-reducing 1, 4-linked alpha-Dglucose residues with releasing of alpha-D-glucose. Treating the adults of E. integriceps by different concentrations of A. annua extract showed the reduction in the activity of - and -glucosidases so that increasing of plant extract concentrations enhanced the enzyme inhibition that emphasais disruption of consumption rates and food conversion efficiencies (Zibaee & Bandani, 2010a). Hemmingi & Lindroth (1999, 2000) determined the effect of phenolic components on gypsy moth (Lepidoptera, Lymantriidae) and forest tent caterpillar (Lepidoptera, Lasiocampidae), founding reduction of the glucosidase activities in both treated larvae. lipases (EC 3.1.1) are enzymes that preferentially hydrolyze the outer links of fat molecules and have been studied in few insects. Although, there a few studies on insect digestive lipases but the enzyme activity significantly changes due to using botanical insecticides. Senthil Nathan et al. (2006) showed that treating Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae), the rice leaffolder, with Btk, NSKE and VNLE (azadirachtin and neem components) sharply decreased the activity level of lipase in the midgut. Zibaee et al. (2008b) found inhibition of lipase activity in the midgut of Chilo suppressalis Walker (Lepidoptera: Pyralidae) when they add A. annua extract to enzyme samples in vitro. Zibaee & Bandani (2010a) found similar results when adults of E. integriceps fed on food containing A. annua extract.

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Botanical Insecticides and Their Effects on Insect Biochemistry and Immunity

Proteases have a crucial role in food digestion by insects. Different types of proteases are necessary to do this because the amino acid residues vary along the peptide chain (Terra & Ferriera, 2005). There are three subclasses of proteinases involved in digestion classified according to their active site group (and hence by their mechanism): serine, cysteine, and aspartic proteinases. The oligopeptides resulting from proteinase action are attacked from the N-terminal end by aminopeptidases and from the C-terminal end by carboxypeptidases. Studies by Johnson et al. (1990), Senthil-Nathan et al. (2006) and Zibaee and Bandani (2010a) inferred that Botanical insecticides may interfere with the production of certain types of proteases and disable them to digest ingested proteins. Zibaee et al. (2010) investigated the sole and combined effect of A. annua and Lavandula stoechas on digestive enzyme activity in Hyphantria cunea Drury (Lepidoptera: Arctiidae) (Table 1 and 2). A. annua treatment decreased digestive enzyme activities in larvae feed on both mulberry and sycamore in a dos-related manner. Also, treatment of leaves by L. stoechas demonstrated a slightly decrease on digestive enzymes except for protease and lipase. However, the effect of L. stoechas extracts on enzyme activities on sycamore was more with regard to mulberry. Treatment (%) Control 10 15 25

Esterase α-naphtyl 3.76±0.062a 4.11±0.021b 4.27±0.083b 4.75±0.095c

β-naphtyl 3.34±0.035a 3.50±0.012a 3.86±0.048ab 4.22±0.055b

Glutathuion S-transferase CDNB 2.82±0.036a 2.85±0.022b 3.17±0.031bc 3.49±0.025c

DCNB 2.84±0.036a 2.92±0.022b 3.27±0.031bc 3.45±0.025c

Acethylcholinesterase

Alkaline phosphatase

Acid phosphatase

7.56±0.027a 7.32±0.020b 6.01±0.052c 5.31±0.031d

4.92±0.024a 4.91±0.020b 5.11±0.034c 5.46±0.027c

3.93±0.018a 3.88±0.046b 4.15±0.026c 4.35±0.021c

Table 1. Effect of A. annua extract on detoxifying enzyme of E. integriceps hemolymph after 24 h. Zibaee & Bandani (2010a) performed analysis of Lineweaver-Burk plots to provide information regarding the mode of action of A. annua extract against E. integriceps digestive enzymes. In the majority of enzymes, the presence of the plant extract decreased the value of Vmax and increased Km. Since Km has an inverse relationship with the substrate concentration required to saturate the active sites of the enzyme, this indicates decreased enzyme affinity for the substrate (Wilson & Goulding, 1986). In other words, Km is the measurement of the stability of the enzyme-substrate complex and a high Km would indicate weak binding while a low Km would indicate strong binding (Stryer, 1995). The effect of A. annua extract on Vmax showed that it interferes with the rate of break down of the enzyme-substrate complex. Thus, the plant extracts inhibit the enzymes by increasing Km and decreasing affinity of the enzyme to substrate. Plant extracts also diminished the Vmax value, which further indicates that they interfered with the rate of breakdown of the enzyme-substrate complex (Morris, 1978). These results showed a mixed inhibition of plant extract on the enzyme activities of the Sunn pest. In this type of inhibition, plant extracts can bind to the enzyme at the same time as the enzyme binds to the substrate, and this binding affects the binding of the substrate and vice versa (Stryer, 1995; Zibaee and Bandani, 2010a). Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect, where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e. tertiary structure e or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced (Morris, 1978; Stryer, 1995; Zibaee and Bandani, 2010a).


58 Treatment1 Control LD10 LD30 LD50

α-amylase Mulberry Sycamore 1.87±0.09a 1.75±0.28a 1.44±0.05b 1.69±0.05a 1.13±0.00c 1.19±0.04ab 0.84±0.06c 0.99±0.03b

α-Glucosidase Mulberry Sycamore 2.05±0.54a 1.90±0.4a 1.39±0.10b 1.55±0.27b 1.24±0.28b ±0.950.09c 0.52±0.19c 0.14±0.08d

β-Glucosidase Protease Mulberry Sycamore Mulberry Sycamore 3.88±1.03a 2.67±0.28a 3.80±0.00a 2.36±0.00a 2.48±0.07c 2.50±0.39b 3.16±0.00ab 1.62±0.00ab 1.39±0.14c 1.55±0.21c 1.88±0.00b 0.80±0.00b 0.28±0.49d 1.25±0.31d 0.76±0.00c 0.50±0.00c

Lipase Mulberry Sycamore 3.43±0.00a 3.34±0.00a 2.79±0.00b 2.61±0.00ab 2.00±0.00c 2.01±0.00b 1.47±0.00d 0.56±0.00c

1. Concentrations of plant extract are 0.09, 0.22 and 0.42 on mulberry and 0.13, 0.28 and 0.48 on sycamore as . LD10, LD30 and LD50. 2. Means (SEM±) followed by the same letters above bars indicate no significant difference (p < 0.05) according to the Tukey test.

Table 3. Effect of Artemisia annua extract on the digestive enzymes profile (µmol/min/mg protein) of Hyphantaria cunea larvae in the presence of two different host. Treatment1 Control LD10 LD30 LD50

α-amylase α-Glucosidase β-Glucosidase Mulberry Sycamore Mulberry Sycamore Mulberry Sycamore 2.08±0.01a 1.97±0.03a 2.20±0.20a 1.49±0.91a 2.89±0.33a 2.61±0.21a 2.05±0.03a 1.61±0.02b 1.77±0.65b 1.62±0.23 2.42±0.68a 2.46±0.48a 1.89±0.03b 1.38±0.08b 2.37±0.74a 1.53±0.30a 2.71±0.12a 1.79±0.70a 1.71±0.02b 1.11±0.05c 2.15±0.75a 1.54±0.34a 2.45±0.23a 1.41±0.23b

Protease Mulberry Sycamore 3.64±0.00a 3.51±0.00a 3.65±0.00a 3.43±0.00a 3.42±0.00a 3.20±0.00a 3.39±0.00a 3.25±0.00a

Lipase Mulberry Sycamore 3.25±0.00a 2.77±0.02a 3.18±0.00a 2.49±0.00a 2.91±0.00a 2.42±0.00a 2.69±0.00a 2.38±0.00a

1. Concentrations of plant extract are 0.02, 0.11 and 0.32 on mulberry and 0.13, 0.38 and 0.79 on sycamore as . LD10, LD30 and LD50. 2. Means (SEM±) followed by the same letters above bars indicate no significant difference (p < 0.05) according to the Tukey test.

Table 4. Effect of Lavandula stoechas extract on the digestive enzymes profile (µmol/min/mg protein) of Hyphantaria cunea larvae in the presence of two different host.

3. Botanical insecticides and detoxifying enzymes Four types of detoxifying enzymes have been found to react against botanical insecticides including general esterases (EST), glutathione S-transferase (GST) and phosphatases. Esterase (EST) is an important detoxifying enzyme in vivo which hydrolyzes the esteric bond in synthetic chemicals. Also, esterase is one of the enzymes showing the strongest reaction to environmental stimulation (Hemingway & Karunatne 1998). The responses of EST to botanical insecticides were significantly due to using different concentrations of extract and long exposure. In the early stage, plant extract stimulated the expression of EST body to increase the detoxification ability (Zibaee and Bandani, 2010b). In the late stage, because of a toxic effect and time EST activity was suppressed. Glutathione S-transferases (GST) are the mainly cytosolic enzymes that catalyze the conjugation of electrophile molecules with reduced glutathione (GSH), potentially toxic substances become more water soluble and generally less toxic (Grant and Matsumura 1989). GSTs play an important role in insecticide resistance and are involved in the metabolism of organophosphorus and organochlorine compounds (Zibaee et al., 2009b). Other xenobiotics such as plant defence allelochemicals against phytophagous insects induce GST activity (Yu, 1982; Vanhaelen et al. 2001). By treating A. annua extracts on E. integriceps adults, Zibaee and Bandani (2010) reported that activity level of GST in 24 h posttreatment increased significantly for both substrates (CDNB, DCNB) of the enzyme. Its (two or one) activity was dose-dependent and increased by exposuring higher concentration of plant extract. Vanhaelen et al. (2001) showed that Brassicacea secondary metabolites induced GST activity in Myzus persicae and several Lepidopteran species such as Heliothis virescens Fabricius, Trichoplusia ni Hubner and Anticarsia gemmatalis Hubner. The influence of plant


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allelochemicals on GST activity is not limited to the herbivores and was observed in several predators, too (Francis et al. 2000). Alkaline phosphatase (ALP, E.C.3.1.3.1) and acid phosphatase (ACP, E.C.3.1.3.2) are the hydrolytic enzymes, which hydrolyze phosphomonoesters under alkaline or acid conditions, respectively. ALP is primarily found in the intestinal epithelium of animals and its major function is to provide phosphate ions from mononucleotide and ribonucleoproteins for a variety of metabolic processes. ALP is involved in the transphosphorylation reaction and the midgut has the highest ALP and ACP activity as compared to other tissues (Sakharov et al. 1989). The overall activity of ALP and ACP decreased due to increasing of plant extract concentrations so that there were significant differences among control and three treatments. These findings coincided with other reports of plant extract treatments of insects. For example, Senthil Nathan (2006) showed that treatment of rice plants with Melia azedarach Juss (Meliaceae) extracts decreased the activity level of ALP in Cnaphalcrocis medinalis (Guenee). These authors reported that feeding Spodoptera litura Fabricius (Lepidoptera: Noctuidae) on Ricinus communis L. treated with azadirachtin decreases the amount of this enzyme in the midgut (Senthil Nathan & Kalaivani 2005). Changes in ALP and ACP activities after treatment with A. annua extract indicating changes of the physiological balance in the midgut.

4. Botanical insecticides and acethylcholine esterase (AChI) AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter, acetylcholine, in the nervous system of various organisms (Oehmichen & Besserer 1982; Grundy & Still 1985; Wang et al. 2004). Zibaee and Bandani (2010b) demonstrated that A. annua extract inhibited the AChE activity in higher doses which coincided with other reports about effect of botanical insecticides on AChE inhibition. The alteration of AChE was observed in the cockroach, Periplaneta americana L., at 4 ppm of AZA, (Shafeek et al. 2004) and the snail, Limnaea acuminate Lamarck, at 40% and 80% concentrations of neem oil (Singh & Singh 2000). It was also observed that 25 g of distilled water extracts of the botanicals Punica granatum L., Thymus vulgaris L., and Artemisia absinthium L., significantly inhibited the AChE activity of nematodes at 100% concentrations (Korayem et al. 1993). Senthil Nathan et al. (2008) demonstrated that LC50 concentrations of AZA significantly inhibited the activity of AChE compared with control.

5. Botanical insecticides and insect immunity 5.1 Introduction Similar to vertebrates, insects have a capable immunity against microbial infections exposing in their environment. This immunity based on involved components known as cellular and humeral defenses (Beckage, 2008). Cellular immunity consists phagocytosis of aggressive microorganisms by hemocytes, nodule formation and encapsulation. Humoral responses comprises factors related to the recognition of invading microorganisms, melanization and coagulation as well as killing factors such as antimicrobial peptides (AMPs), reactive oxygen species and reactive nitrogen intermediates, including nitric oxide, prostaglandins and eicosanoids (Boman, 1998; Stanley, 2006; Beckage, 2008). Mentioned immune reactions are initiated by pattern recognition molecules allowing insects to distinguish self-components from nonself-ones. Studies have been identified specific

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pattern recognition receptors responding to components in microorganisms such as peptidoglycans and lipopolysaccharides that are main compounds in the cell walls of bacteria and fungi (Theopold et al., 1999; Dziarski, 2004). Peptidoglycan recognition proteins (PGRPs) have been identified in several insect species as activating cascade of melanization on invasive microorganisms (Rolff & Reynolds, 2010). There are specific PGRPs for grampositive, gram-negative and fungi in the hemolymph of insects. Two signaling pathways namely Toll and Imd have been activated after recognition of gram-positive microorganisms and fungi as well as gram-negative ones, respectively (Rolff & Reynolds, 2010). These signaling pathways lead to activation of cellular immunity and antimicrobial peptides via final Dif and Relish molecules in nucleus of hemocytes (Tzou et al., 2002; Leihl et al., 2006). Different environmental factors can definitely affect immune reactions of insects that elucidation of these factors is a significant part to clarify various aspects of these mechanisms. Temperature, different ions and insecticides are some of the most important affecting factors (Zibaee et al., 2009c). In agriculture, combined tactics (as Integrated Pest management) are used to obtain efficient control of insects by considering the lowest disruption in environment. Several studies have been conducted to find combined effect of insecticides, highlighted by botanical materials, and microbial agents on insects. Results revealed that botanical compounds decrease immune ability of insects against microbial agents that describes in forward sections. 5.2 Effect of botanical insecticides on morphology, number of hemocytes and Phagocytosis In insect immunity, circulating hemocytes have crucial roles in both cellular mechanisms and producing antimicrobial components. Five basic types of hemocytes have been identified as prohemocytes, plasmatocytes, granulocytes, adipohemocytes and oenocytoids (Lavine & Strand, 2002). Prohemocytes as the smallest one are the basic hemocytes that developed to plasmatocytes and granulocytes when an infectious challenge appeared in the hemolyph. They recognized as large central nucleus and narrow cytoplasm (Lavine & Strand, 2002; Zibaee & Bandani, 2010a) (Figure 2a). Plasmatocytes and granulocytes are the important hemocytes in immune responses to pathogens via phagocytosis (Granulocytes and relatively Plasmatocytes), nodule formation and encapsulation (Strand, 2008). They discriminate each other by spindle shape of plasmatocytes and rounded granulus granulocytes (Figure 2b and c). Adipohemocytes contain lipid droplets so some literature consider them as fat bodies instead of hemocyte (Figure 2d) (Beckage, 2008). Oenocytoids have two specific shape based on intact and immune challenged insects. In normal situation, oenocytoids are spherical cells with peripheral nucleus and crystalline inclusions without any granules (Figure 2e). When an immune challenge occurred, nucleus is going to be smaller and granules appear showing their crucial roles in phenoloxidase1 (PO) cascade (Strand, 2008; Beckage, 2008). Different environmental factors could affect insect hemocytes both morphologically and functionally. For example, elevation of environmental temperature increases numbers of plasmatocytes and granulocytes up to 30-40 °C in addition their nodulation ability (Zibaee et al., 2009). Also, different divalent cations have positive effect on hemocytes to provide a cellular network entrapping pathogens in the hemolymph (Willot et al., 2002; Willot and Tran, 2002; Zibaee et al., 2009c) (Figure 3).

1 Phenoloxidases have crucial role in immune recognition pathways and melanization of nodules and capsules around an pathogens.


(a)

(b)

(c)

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(d)

(e)

Fig. 2. (a-e) Light microscopy of E. integriceps hemocytes. (a) A prohemocyte with a large nucleus (thin arrow) and a thin cytoplasm (b) A plasmatocyte exhibiting a spindle shape and cytoplasmic elaborations. (c) A granulocyte filled with the typical granules in the cytoplasm (arrow) and large nucleus (arrow). (d) An adipohemocyte with lipid droplets spreading in the cytoplasm and specific cytoplasm elaborations (e). An oenocytoid with a round eccentric nucleus and crystalin inclusions. Magnification 40X with the exception of (b) (60X). Bar= 50 µm, with the exception of (b), 33 µm. (Zibaee, A., Bandani, A. R., TalaeiHassanlouei, R. & Malagoli, D. et al., Unpublished data).


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Fig. 3. Phase contrast microscopy of plasmatocytes incubated 12 h by calcium. (a) Control plasmatocytes without incubation by calcium. (b) plasmatocytes incubated by 5 mM concentration of calcium (Zibaee et al., 2009c; Entomological Research; Wiley-Blackwell publishing). In addition of these positive factors on hemocytes of insects, several other factors, mainly insecticides, have negative effects on number and morphology of them (Figure 4). There are some reports on effects of plant products on the hemocytes such as Periplaneta americana L. (Blattodea: Blattidae) (Qadri & Narsaiah, 1978), Dysdercus koenigii Fabricius (Hemiptera: Pyrrhoeoridae) (Saxena & Tikku, 1990; Tikku et al., 1992), Cyrtacanthacris tatarica L.

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(Orthoptera: Acrididae) (Peter & Ananthakrishnan, 1995) and Spodoptera litura Fabricius (Lepidoptera: Noctuidae) (Sharma et al., 2001, 2003, 2008). Studies by scan electron microscopy demonstrated the complete loss of filopods in plasmatocytes and cytoplasmic projections in granular hemocytes of S. litura larvae treated with Neem gold (Sharma et al., 2003). Sharma et al. (2008) also find similar results on effect Artemisia calamus oil on larvae of S. litura as loss of cytoplasmic projections in granular hemocytes. Interestingly, they observed vacuolization in the cytoplasm and degeneration of the organelles, both in plasmatocytes and granular hemocytes (Sharma et al., 2003). So, it was concluded that rapid degeneration of granular hemocytes, initiated by vacuolization and loss of firmness of organelles leading to degranulation and a degenerative transformation within a period of 48 h, subsequently resulting the total collapse of immunity-building mechanism of S. litura (Sharma et al., 2008). Atemisia annua extract altered number of hemocytes and their phagocytic activity in Eurygaster integriceps Puton (Hemiptera: Scutelleridae). Zibaee & Bandani (2010a) reported that treatment of E. integriceps with A. annua extract affected the total number of hemocytes circulating in the hemolymph indicating that the responses could be due to the toxic effect on the immune cells reducing number of hemocytes attached to fungal spores. Meanwile, an extremely low phagocytic activity was observed in these bioassay experimental groups. Since the attachment of fungal spores to the hemocyte surface is an essential prerequisite to the triggering of phagocytic responses, suggesting that the cellular activity or recognition of spore by hemocyte receptors may be compromised in the hemocytes of insects treated with A. annua. Phagocytosis of microbial cells involves interactions between lections on phagocytic cells and sugars on microbial surfaces (Nayar & Knight, 1997). Since A. annua extracts suppress phagocytosis (and also nodule formation and PO activity) at different concentrations, this suggests that it may interfere with the ligend-receptor interactions that are likely to occur at the plasma membrane of specific hemocytes because the majority of interactions between cellular and humeral components of the insect immune system are receptor-mediated (Ratcliffe & Rowley, 1987). Therefore, plant extracts at the sub-lethal levels might be enough to interfere with the function of specific receptors, e.g. b-1,3-glucanspecific protein of many insect-species hemocytes, or cause ultrastructural alteration which interfere with normal hemocyte function (Vey et al., 2002). Garcia et al. (2006) reported significantly higher numbers of Trypanosoma rangeli in the hemolymph of Rodnius prolixus L. (Hemiptera: Reduviidae) fed on blood containing physalin B at days 2, 4, and 6 post-injection in contrast to that observed in the control. In fact, their data supported the hypothesis that physalin B is an immunomodulator to T. rangeli challenge in R. prolixus. They concluded four main points for verifying this hypothesis. (i) Mortality of R. prolixus in response to common parasite challenge was expressed in a concentration-dependent way in insects treated with concentrations ranging from 0.01 to 1 µg of physalin B/ml of blood meal the idea was supported by Zibaee & Bandani (2010a). (ii) The death rate was significantly enhanced in insects that received concentrations of 0.1 and 1 µg of physalin B and were infected with flagellates. (iii), the hemocyte microaggregation response and nitrite/nitrate concentration, which represent metabolic products of nitric oxide reactions and RNI metabolism against T. rangeli infection, was significantly reduced in the hemolymph of insects treated with physalin B (0.1 µg /ml) when compared with infected untreated controls. (iv) The number of parasites in the hemolymph of treated-insects was significantly higher than that observed in insects feeding on blood without physalin B. Based on these results, they proved that physalin B is a


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regulator of microaggregation and nitric oxide reactions to parasite challenge in 5th-instar R. prolixus larvae (Garcia et al., 2006). 5.3 Effect of botanical insecticides on nodule formation and phenoloxidase activity PO enzymes in hemolymph that have tyrosinase-like activity can hydroxylate tyrosine (EC 1.14.18.1) and also can oxidize o-diphenols to quinones (EC 1.10.3.1) (Gorman et al., 2007) so called o-phenoloxidases. The quinones produced by PO undergo a series of additional enzymatic and non-enzymatic reactions leading to polymerization and melanin synthesis in the final stages of nodulation and encapsulation against invading microorganisms (Zibaee et al., 2011). In fact, insect PO are synthesized as zymogens called pro PO which are activated by proteolytic cleavage at a specific site in response to infection or wounding (Cerenius & Söderhall, 2004). Active PO catalyzes the formation of quinones, which undergoes further reactions to form melanin (Cerenius and Söderhäll, 2004; Gorman et al., 2007). Zibaee & Bandani (2010a) showed the negative effect of A. annua extract on nodule formation and phenoloxidase activity of E. integriceps (table 2 and 3). Lineweaver-Burk plots analysis of PO activity after treating insects by plant extract revealed an inhibition on enzyme activity by decreasing Vmax value and increasing Km one. Since the Km has an inverse relationship with the substrate concentration required to saturate the active sites of the enzyme, this indicates decreased enzyme affinity for substrate (Zibaee et al., 2011). In other words, Km is the measurement of the stability of the enzyme-substrate complex and a high Km would indicate weak binding and a low Km strong binding. The effect of A. annua extract on the Vmax shows that it interferes with the rate of break-down of the enzyme-substrate complex. Thus, plant extract inhibit the enzymes by increasing the Km and decreasing affinity of the enzyme to substrate. Plant extract also diminished the Vmax value which further indicates that they interfere with the rate of breakdown of the enzyme-substrate complex. These results showed a mixed inhibition of plant extract on the enzyme activities of the Sunn pest. In this type of inhibition, plant extract can bind to the enzyme at the same time as the enzyme binds to substrate and this binding affects the binding of the substrate, and vice versa. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure e or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced (Zibaee et al., 2011).

6. Acknowledgement The author would like to thank Dr. Ali R. Bandani for his great contribution to provide many relevant experiments especially on immunity. It is my especial appreciation to my wife, Samar Ramzi (Entomology MSc) for her assistance in re-read the text and better presentation of the chapter.

7. References Akhtar, Y. & Isman, M. B. (2004) Comparative growth inhibitory and antifeedant effects of plant extract and pure allelochemicals on some phytophagous insect species, Journal of Applied Entomology 128, 32–38. Beckage, N. E., 2008. Insect Immunology. Academic press. 348 pp.

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Qadri, S.S.H., Narsaiah, J., 1978. Effect of azadirachtin on the moulting processes of last instar nymphs of Periplaneta americana (L.). Indian J. Exp. Biol. 16, 1141–1143. Ratcliffe, N.A., & Rowley, A.F. (1987) Insect response to parasites and other pathogens. pp. 271–332 in Soulsby, E.J.L. (Ed.) Immunology, Immunoprophylaxis and Immunotherapy of Parasitic Infections. Boca Raton, FL, USA, CRC Press. Rolff, J. and Reynolds, S. E. 2010. Insect infection and immunity (evolution, Rcology and Mechanisms). Oxford university press. 254 pp. Saleem, M. A. & Shakoori, A. R. (1987) Point effects of Dimilin and Ambush on enzyme activies of Tribolium castaneum larvae. Pesticide Biochemistry and Physiology 29, 127–137. Saxena, B.P., Tikku, K., 1990. Effect of plumbagin on hemocytes of Dysdercus koenigii F. Proc. Indian Acad. Sci. (Anim. Sci.) 99 (2), 119–124. Schmutterer, H. (2002) The Neem Tree. Mumbai: Neem Found. 892 pp. Scott, I. M., Jensen, H., Scott, J. G., Isman, M. B., Arnason, J. T., Philogène, B. J. R. 2003. Botanical Insecticides for Controlling Agricultural Pests: Piperamides and the Colorado Potato Beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Archives of Insect Biochemistry and Physiology 54: 212–225. Senthil Nathan, S., Chunga, P.G. & Muruganb, K. (2006) Combined effect of biopesticides on the digestive enzymatic profiles of Cnaphalocrocis medinalis (Guenee) (the rice leaffolder) (Insecta: Lepidoptera: Pyralidae). Ecotoxicology and Environmental Safety 64, 382–389. Sharma, P. R., Sharma, O. P. and Saxena, B. P. 2008. Effect of sweet flag rhizome oil (Acorus calamus) on hemogram and ultrastructure of hemocytes of the tobacco armyworm, Spodoptera litura (Lepidoptera: Noctuidae). Micron 39 (2008) 544–551. Sharma, P.R., Sharma, O.P., Saxena, B.P., 2001. Ultrastructure of the haemocytes of the tobacco armyworm, Spodoptera litura Fab. (Lerpidoptera; Noctuidae). Biol. Bratislava 56 (3), 277–285. Sharma, P.R., Sharma, O.P., Saxena, B.P., 2003. Effect of neem gold on haemocytes of the tobacco armyworm, Spodoptera litura (Fabricius) (Lepidoptera; Noctuidae). Curr. Sci. 84 (5), 690–695. Shekari, M., Jalali Sendi, J., Etebari, K., Zibaee, A. & Shadparvar, A. (2008) Effects of Artemisia annua L. (Asteracea) on nutritional physiology and enzyme activities of elm leaf beetle,Xanthogaleruca luteola Mull (Coleoptera: Chrysomellidae). Pesticide, Biochemistry and Physiology 91, 66-74. Stanley, D. W. (2006a). Prostaglandins and other eicosanoids in insects: Biological significance. Annu. Rev. Entomol. 51, 25–44. Strand, M. (2008) The insect cellular immune response. Insect Science 15: 1-14. Terra, W. R., Ferriera, C., 2005. Biochemistry of digestion. In: Comprehensive molecular insect science by Lawrence I. Gilbert, Kostas Iatrou, and Sarjeet S. Gill, volum 3. Elsevier. Pp 171-224. Theopold, U., Rissler, M., Fabbri, M., Schmidt, O. & Natori, S. (1999) Insect glycobiology: a lectin multigene family in Drosophila melanogaster. Biochemistry Biophysics Research Community 261, 923-927. Tikku, K., Saxena, B.P., Satti, N.K., Suri, K.A., 1992. Plumbagin-induced ultrastructural haemocytic response of Dysdercus koenigii (F.). Insect Sci. Appl. 13 (6), 787–791.

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Tzou, P., Reichhart, J.M., and Lemaitre, B. (2002) Constitutive expression of a single antimicrobial peptide can restore wild-type resistance to infection in immuno-defi cient Drosophila mutants. Proceedings of the National Academy of Sciences USA 99, 2152–2157. Vey, A., Matha, V. & Dumas, C. (2002) Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction. Journal of Invertebrate Pathology 80, 177–187. Willot, E. and Tran, H. Q. 2002. Zinc and Manduca sexta hemocyte functions. Journal of Insect Science. 2.6. Available online: insectscience.org/2.6. Willott E, Hallberg CA, Tran HQ (2002) Influence of Ca2+ on Manduca sexta Plasmatocyte Spreading and Network Formation. Archives of Insect Biochemistry an Physiology 49: 187– 202. Zibaee, A. & Bandani, A. R. (2010a) Effects of Artemisia annua L. (Asteracea) on digestive enzymes profiles and cellular immune reactions of sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauvaria bassiana. Bulletin of Entomological Research 100, 185-196. Zibaee, A., Bandani, A. R. & Ramzi, S. (2008b) Lipase and invertase activities in midgut and salivary glands of Chilo suppressalis (Walker) (Lepidoptera, Pyralidae), rice striped stem borer. Invertebrate Survival Journal 5, 180-189. Zibaee, A., Bandani, A. R. & Ramzi, S. (2009a) Characterization of α and β-glucosidases in midgut and salivary glands of Chilo suppressalis Walker (Lepidoptera: Pyralidae), rice striped stem borer. Comptes Rendus Biologies 332, 633–641. Zibaee, A., Bandani, A. R., Talaei-Hassanlouei, R. & Malagoli, D. (2009c) Temperature and Ca2 +ion as modulators in cellular immunity of the Sunn pest Eurygaster integriceps Puton (Heteroptera: Scutelleridae). Entomological Research 39, 364–371. Zibaee, A., Bandani, A. R., Kafil, M. & Ramzi, S. (2008a) Characterization of α-amylase in midgut and salivary glands of Chilo suppressalis Walker (Lepidoptera: Pyralidae), rice striped stem borer. Journal of Asia-Pacific Entomology. 11, 201-205. Zibaee, A. & Bandani, A. R. (2010b) A study on the toxicity of the medicinal plant, Artemisia annua L. (AStracea) extracts the Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae). Journal of Plant Protection Research 50, 48-54. Zibaee, A., Sendi, J., Alinia, F., Ghadamyari, M. & Etebari, K. (2009b) Diazinon resistance in different selected strains of Chilo suppressalis Walker (Lepidoptera: Pyralidae), rice striped stem borer, in the north of Iran. Journal of Economic Entomology 102, 11891196. Zibaee, I., Bandani, A. R., Sendi, J. J., Talaei-Hassanlouei, R. & Kouchaki, B. (2010) Effects of Bacillus thurengiensis var. kurstaki, and medicinal plants (Artemisia annua L.) and (Lavandula stoechas L.) extracts on digestive enzymes and Lactate dehydrogenase of Hyphantria cunea Drury (Lepidoptera: Arctiidae). Invertebrate Survival Journal. 7: 251261. Zibaee, A., Bandani, A. R. & Malagoli, D. (2011) Purification and characterization of phenoloxidase from the hemocytes of Eurygaster integriceps (Hemiptera: Scutelleridae). Comparative Biochemistry and Physiology Part B. 158: 117-123.

5 The Production, Separation and Stability of Pyoluteorin: A Biological Pesticide Wei Wang, Hui Dong, Jingfang Zhang, Yuquan Xu and Xuehong Zhang

Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China

1. Introduction Pyoluteorin (Plt, 4,5-dichloropyrrol-2-yl 2,6-dihydroxyphenyl), a polyketide antibiotic produced by certain strains of Pseudomonas spp., including the rhizobacteria Pseudomonas fluorescens CHA0 (Maurhofer et al., 1992), Pseudomonas fluorescens Pf-5 (Corbell & Loper, 1995), Pseudomonas fluorescens S272 (Yuan et al., 1998) and Pseudomonas sp. M18 (Hu et al., 2005) et al., was first identified and separated by Takeda from Pseudomonas aeruginosa (Tekeda, 1958). Plt is a yellow crystal composed with a bichlorinated pyrrole linked to a resorcinol moiety (see Fig. 1), it can be completely dissolved in organic solvents such as methanol and chloroform (Wang et al., 2008). Plt can effectively inhibit phytopathogen fungi, including the plant pathogen Pythium ultimum, and suppress plant diseases caused by this fungus (Howell & Stipanovic, 1980; Maurhofer et al., 1992; Maurhofer et al., 1994). Moreover, in some instances, it contributes to the ecological competence of the producing strain within Rhizosphere (Howell & Stipanovic, 1980; Carmi et al., 1994; Yuan et al., 1998; Babalola, 2010).

Cl

OH

HO

O

N H

Cl

Fig. 1. Chemical structure of Plt Many studies have focused on the metabolic regulation of Plt biosynthesis to enhance Plt production in cell cultivation. It was reported that the transcriptional activator gene pltR linked to Plt biosynthetic genes is required for Plt production (Nowak-Thompson et al., 1999). Whereas the regulator gene pltZ, which is located downstream of the Plt gene cluster in the genome of Pseudomonas sp. M18, could repress Plt production (Huang et al., 2004). Plt biosynthesis could be enhanced by the amplification of the housekeeping sigma factor rpoD in Pseudomonas fluorescens CHA0 (Schnider et al., 1995). The two component regulatory system GacS/GacA could positively regulate Plt production (Laville et al., 1992; Whistler et

70


al., 1998), and inactivation of rpoS (Sarniguet et al., 1995) or lon (Whistler et al., 2000) also resulted in the overproduction of Plt in Pseudomonas fluorescens Pf-5. The culture conditions are usually important to the yield of any fermentation product. Carbon and nitrogen sources generally play a significant role because these nutrients are directly linked to cell proliferation and metabolite biosynthesis (Park et al., 2001; Casas Lopez et al., 2003; Li et al., 2008). In relation to this, Yuan et al. (Yuan et al., 1998) reported ethanol as a sole carbon source for Plt production by Pseudomonas fluorescens S272 cultivation in shake flasks. Duffy and Defago’s study showed that Plt production was stimulated by glycerol but was repressed by glucose in cell culture of Pseudomonas fluorescens CHA0 (Duffy & Defago, 1999). The influence of other environmental factors on Plt production was also investigated by many researchers. In Brodhagen’s study, Plt was found to be induced by itself in cell culture of Pseudomonas fluorescens Pf-5 (Brodhagen et al., 2004). In Pseudomonas fluorescens S272 cultivation, high NaCl concentration or heat shock could increase the production of Plt (Nakata et al., 1999). Chloride could also increase Plt biosynthesis in cell culture of Pseudomonas fluorescens YGJ3 (Matano et al., 2010). The minerals, such as zinc, could enhance Plt production in cell cultivation of both Pseudomonas fluorescens CHA0 (Duffy & Defago, 1999) and Pseudomonas fluorescens 4-92 (Saikia et al., 2009). However, there have been no reports on the effect of carbon and nitrogen ratio on Plt production through the fermentation of Pseudomonas spp. and no work was done on the scale-up fermentation for Plt production. As a potential biological pesticide, understanding the stability of Plt under different environmental conditions as well as its residue in soil after incorporation into the environment was great important for its commercial use. Some analytical methods of Plt from the fermentation broth based on HPLC have been reported (de Souza & Raaijmakers, 2003; La Fuente et al., 2004). Kim et al. described a method for quantification analysis of Plt in fermentation broth with liquid chromatography–mass spectrometric (LC–MS) (Kim et al., 2003). Wang et al. developed a succinct quantitative method of capillary zone electrophoresis (CZE) for the determination of Plt in fermentation liquor of Pseudomonas sp. M18 (Wang et al., 2005). Trace determination of Plt in soil was done by Dong et al. using capillary electrophoresis (CE) (Dong et al., 2011). The degradation of Plt in water under different pH, temperature and light sources were studied by Zhang et al. (Zhang et al., 2010). Pseudomonas sp. M18 is one of the plant growth promoting rhizobacteria (PGPR) selected in our lab, which can produce the secondary antifungal metabolites of Plt to suppress the root diseases caused by the soil-borne phytopathogens of crop plants (Hu et al., 2005). The chromosomally rsmA-inactivated and NTG mutation strain of Pseudomonas sp. M18R is a high Plt production strain obtained in our laboratory (Zhang et al., 2005). The identification of an effective medium formulation for Plt production is of great importance because it usually plays a pivotal role in cell growth and the production of metabolites. As a highly efficient anti-fungal metabolite, it is also important to develop the separation and purification method of Plt from its fermentation broth for large-scale preparation. The medium optimization studies described in the literature have been conducted either by the “one-variable-at-a-time” technique or the “response surface method” (RSM) and “central composite design” (CCD) approach (Chang et al., 2002; Li et al., 2008). In this paper, the carbon or nitrogen sources and their initial concentrations were studied using the “one-variable-at-atime” method for Plt production from Pseudomonas sp.M18R. Central composite design and response surface analysis were then applied to determine the optimal carbon/nitrogen ratio for Plt production. Influence of mineral amendment on Plt production was also investigated,

The Production, Separation and Stability of Pyoluteorin: A Biological Pesticide

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and the optimized medium was verified in both shake flask and bioreactor cultivation. The separation and purification method for Plt from the fermentation broth of M18R was investigated. The stability of Plt under different conditions were studied (Zhang et al., 2010), and a sensitive analytical method based on capillary electrophoresis (CE) for studying the degradation of trace amounts of Plt in soil samples was developed (Dong et al., 2011). The information obtained is considered fundamental and useful for the development of several Pseudomonas strain cultivation processes for efficient large-scale Plt production.

2. Materials and methods 2.1 Bacterial strain and seed culture conditions The chromosomally rsmA-inactivated mutant strain of Pseudomonas sp.M18R was obtained as described by Zhang et al. (Zhang et al., 2005). The seed culture medium (with an initial pH of 7.2) consisted of the following components: glycerol, 18 g/L; peptone, 20 g/L; K2HPO4·3H2O, 0.732 g/L; and MgSO4, 0.514 g/L. The stock culture was maintained on agar slants, which were inoculated with M18R, incubated at 28℃ for 12 hours, and then used for seed culture inoculation. For the seed culture, about 5 mm2 of the M18R bacterial slants was punched out with a sterilized cutter and was then transferred to a 250 mL Erlenmeyer flask containing 50 mL of the culture medium. The cultivation was maintained at 28°C on a rotary shaker (220 rpm) for 10.5 hours, reaching an OD600 at about 1.1. 2.2 Experiments on M18R fermentation The effects of carbon sources were studied using various carbon sources (18 g/L) such as glycerol, sucrose, glucose, fructose, lactose, maltose, and ethanol. For the investigation of nitrogen sources, 20 g/L of peptone, yeast extract, casein enzymatic hydrolysate, and casein acid hydrolysate were studied. For the investigation of initial carbon concentrations, different glycerol levels were used at 15, 18, 21, and 24 g/L. Furthermore, 12, 16, 20, and 24 g/L peptone was used for the study of initial nitrogen concentrations. In the experiments on the effects of carbon/nitrogen ratios, glycerol and peptone levels in the medium were changed, and a statistical approach was used. For the studies of mineral sources, 0.02 g/L ZnSO4, CuSO4, CoCl2, FeSO4, MnCl2 or NaCl was added individually into the control medium. The cells were harvested on 72 h to analyze Plt production. Inoculation was done by transferring 7.5 mL of the above seed culture broth to a 150 mL fermentation medium in a 500 mL shake flask. The fermentation medium was the same as the seed culture medium except for the conditions studied. The cultivation was conducted at 28°C on a rotary shaker at 220 rpm. Multiple flasks were run under each condition for all cultures. The cultivation data represent the mean values with the standard deviations from three independent flasks. The scale-up fermentation was carried out in a 10 L SY-3000E bioreactor (Shiyou Company, Shanghai, China) with 6 L optimized medium. The fermentation was conducted at an aeration rate of 1.6 vvm and an agitation speed of 250 rpm for 96 h and the shake flask culture was done as control. 2.3 Central composite design RSM was used to optimize the glycerol and peptone ratios for enhanced Plt production based on CCD (Li et al., 2008). The second-order model used to fit the CCD experimental results is shown in Eq. (1):

72


y  0   i xi   ii xi2   ij xi x j

(1)

where y is the predicted response,0 is a constant,i is a coefficient for the linear effect,ii is a coefficient for the quadratic effect,ij is a coefficient for the interaction effect, and xi and xj are the coded levels of variables xi and xj, respectively. The fitness of the second-order model was checked using the adjusted coefficient of determination R2, and its statistical significance was determined by the application of Fischer’s F test. The software programs SAS (version 9.1 by SAS Institute Inc., NC, USA) and MatLab (version 7.1 by Mathworks, Inc.) were used for regression and graphical analyses of the data obtained, respectively. 2.4 Measurement of cell growth and Plt production Cell growth was assayed in terms of optical density at 600 nm (OD600). A 1 mL culture broth was centrifuged at 12000 rpm for 5 min, and the sediments were re-suspended and diluted in distilled water. The analytical procedures for Plt production were slightly modified as described by Zhang et al. (Zhang et al., 2005). For Plt extraction, 1 mL of each culture broth was mixed with the same volume of ethyl acetate. The upper layer was collected after complete agitation and centrifugation at 8000 g for 5 min. The water layer was then extracted once more with 0.5 mL ethyl acetate. The combined extracts were finally dried in a desiccated vacuum at 40°C and then dissolved in 1 mL of HPLC grade methanol. Using a Shimadzu LC-8A HPLC apparatus equipped with a variable-wavelength UV detector (Shimadzu, SPD-8A), 20L sample was analyzed by reverse-phase HPLC. A Zorbax SB-C18 column (250×4.6 mm2; 5m) was used at 25°C. The mobile phase consisted of 70% methanol (vol) and 30% water (vol), with the flow rate kept constant at 1 mL/min. Plt was monitored and quantified at 308 nm, and was then identified by comparison with its authentic sample. 2.5 Plt purification Firstly, the fermentation broth was extracted with ethyl acetate and the crude Plt extract was obtained. Secondly, the crude Plt extract was separated by silica gel column chromatography with a glass column (700×50 mm2) packed with 500 g analytical grade silica of 100-200 mesh. The column was eluted with benzene-acetic acid (20:1,vol) and the elution which contained Plt was collected and evaporated at 40°C to dry, and the residue was dissolved in methanol (HPLC grade) for further purification. Preparative HPLC was conducted for further purification of Plt using a Tigerkin C-18 column (300×20 mm2; 10m) under 25°C with 50% methanol as the mobile phase. The flow rate was kept constant at 15 mL/min and the highest peak containing Plt was collected. Plt crystal was finally obtained after the elution was evaporated at 40°C to dry. 2.6 Trace analysis of Plt in soil samples (Dong et al., 2011) A series of Gly-NaOH buffers with different pH values and concentrations were prepared. The stock solution of internal standard (IS) was prepared by adding 1.0 mg PCA into 10.0 mL methanol to fix its concentration at 100 g/mL. Standard solutions are prepared by dissolving Plt with different concentrations in methanol, forming a concentration gradient of 100, 75, 50, 25, 5, 2, 0.5 g/mL. All solutions and buffers were stored at 4°C. Plt was extracted from soil samples (air dried, mixed and sieved through a 2-mm sieve) with ethanol. The extraction procedure was performed as follows: the working standard solution

The Production, Separation and Stability of Pyoluteorin: A Biological Pesticide

73

was spiked into 10.0 g soil in an appropriate volume, which was ultrasonic extracted with 50 mL ethanol for 20 min. The extraction liquid was collected and the solid sample was transferred to the Soxhlet extractor for 4 h using another batch of 100 mL ethanol. After mixing and evaporating all organic phases, the dry residue was dissolved in 5 mL methanol and then centrifuged at 3000 rpm for 10 min. A portion of the 500 L methanol layer was distributed to one Eppendorf tube with an addition of 125 L PCA (25 g/mL). All solutions were stored at 4°C before injection into capillary. The capillary-based experiments were carried out with an ACS 2000 HPCE apparatus (Beijing Cailu Scientific Inc., Beijing, China). The operating system is Windows XP professional SP2 with a HW-2000 chromatography work station (Qianpu Software Co. Ltd., Nanjing, China). Electrophoresis was performed in an untreated fused-silica capillary of 53 cm total length (44 cm effective length) × 75 m I.D. (375 m O.D.) (Yongnian Optical Fiber Factory, Hebei, China). Before use, a new fused-silica capillary was pressure-rinsed with 0.1 M NaOH for 20 min, ultra-pure water for 20 min, 0.1 M HCl for 20 min, ultra-pure water for 20 min, and CE running buffer for 30 min orderly. The capillary was rinsed with CE running buffer for 5min between injections and finally stored in water when not in use. The samples were injected in pressure mode at the inlet (13 mbar, for 45 s). The ultimate working voltage was 10 kV. Plt degradation study in soil was carried out with samples of both near-surface soil (0~10cm in depth) and rhizosphere soil (10 cm below surface), which were initially spiked with Plt at concentration of 500 g/kg. The extraction and analytical procedure were done as described above. 2.7 Degradation of Plt under different conditions The degradation of Plt in water was done as describe by zhang et al. (Zhang et al., 2010). Plt solutions were prepared using our purified Plt and pure water, with the Plt concentrations varying between 121.5 mg/L and 626.5 mg/L. Temperature was thermostat-controlled, and irradiation was carried out in a Pyrex vessel, using natural sunlight. Samples were taken from the reactor periodically and were analyzed immediately by HPLC. The sample treatment procedure was as follows: 200 L ethyl acetate was added to the reactive solutions to stop the reaction and extract Plt. The organic solution was then dehydrated to obtain a powder residue of Plt. All experiments were preformed in duplicate.

3. Results and discussion 3.1 Optimization of M18R cultivation 3.1.1 Experiments on carbon sources and initial carbon concentrations Carbohydrates are important carbon and energy sources for bacterial growth and metabolite biosynthesis. In the cell culture of Pseudomonas fluorescens S272, a high Plt production titer was obtained using ethanol as sole carbon source (Yuan et al., 1998). In the current work, we investigated the effects of ethanol as well as various common carbon sources, such as glycerol, sucrose, glucose, fructose, lactose, and maltose on M18R cultivation. The time profiles of cell growth (OD600) and Plt production are shown in Fig. 2. It can be seen that the cell grew well in glycerol, glucose, and fructose, and the maximum cell density was obtained in glucose after 72 hours of cultivation. A higher Plt production titer was obtained in glycerol and fructose for M18R. The order of maximum Plt production titer was 540.2±20.0, 401.2±24.8, 270.1±20.1, 212.6±12.6, 119.7±11.7, 37.2±3.2, and 45.7±3.7 mg/L in glycerol, fructose, ethanol, glucose, sucrose, lactose, and maltose media, respectively.


74

600

A

10

OD 600 nm

Plt production (mg/L)

12

8 6 4 2 0 0

20

40

60

80

100

Cultivation time (h)

B

500 400 300 200 100 0 0

20

40

60

80

100


Fig. 2. Time profiles of cell growth (A) and Plt production (B) for the cell culture of P. M18R with different carbon sources (18 g/L). Symbols for different carbons: glycerol (dark diamond), fructose (open diamond), sucrose (dark triangle), maltose (open triangle), ethanol (dark square), glucose (open square), and lactose (dark circle). The error bars in the figure indicate the standard deviations from three independent samples. Initial glycerol concentration (g/L) 15 (72 h)a 18 (72 h) 21 (60 h) 24 (60 h)

Plt production (mg/L) 460.3± 27.8 568.3± 30.1 464.3± 31.9 423.7± 19.8

Plt productivity (mg/L per hour) 6.39± 0.39 7.89± 0.42 7.74± 0.53 7.06± 0.33

Plt yield on carbon (mg/g) 30.7± 1.9 31.6±1.7 22.1± 1.5 17.7± 0.8

Table 1. Effects of initial glycerol concentration on Plt production for P.M18R cultivation. a Cultivation time when the maximum Plt production was achieved. Based on the above results, glycerol was selected as the carbon source for the cell culture of M18R. The effects of initial glycerol concentrations on Plt production are shown in Table 1. The highest production and productivity of Plt were obtained at an initial glycerol concentration of 18 g/L. The Plt production was decreased at high initial glycerol concentrations (21 or 24 g/L), the inhibitory effect of high initial carbon concentration on metabolite biosynthesis was also observed in ganoderic acid biosynthesis by Ganoderma lucidum (Fang & Zhong, 2002). 3.1.2 Experiments on nitrogen sources and initial nitrogen concentrations In the cell culture, both carbon and nitrogen sources are very important for cell growth and metabolite production. Various organic nitrogen, including peptone, yeast extract, casein enzymatic hydrolysate, and casein acid hydrolysate, were studied in this paper. The time profiles of cell growth (OD600) and Plt production for the cell culture of M18R in different nitrogen were shown in Fig. 3. It is evident that the cells grew well in peptone, and a maximum Plt production titre of 553.2±27.2 mg/L was achieved after 72 hours of cultivation in the peptone medium. The effects of initial peptone concentrations on Plt biosynthesis were further examined, the results of which are shown in Table 2. The respective maximum Plt production and productivity of 568.3±30.1 mg/L and 7.89±0.42 mg/L per hour were obtained at an initial peptone concentration of 20 g/L.

75

The Production, Separation and Stability of Pyoluteorin: A Biological Pesticide 10 8

OD 600 nm


600

A

6 4 2 0 0

20

40

60

80

100


B

500 400 300 200 100 0 0

20

40

60

80

100


Fig. 3. Time profiles of cell growth (A) and Plt production (B) for the cell culture of P. M18R with different nitrogen sources (20 g/L). Symbols for different nitrogens: peptone (open square), yeast extract (dark square), casein acid hydrolysate (open triangle), and casein enzymatic hydrolysate (dark triangle). The error bars in the figure indicate the standard deviations from three independent samples. Initial peptone concentration (g/L)


Plt productivity (mg/L per hour)

Plt yield on carbon (mg/g)

12 (84 h)a

393.2± 30.8

4.68± 0.37

21.8± 1.7

16 (72 h)

485.5± 15.0

6.74± 0.21

27.0±0.8

20 (72 h)

568.3± 30.1

7.89± 0.42

31.6±1.7

24 (60 h)

332.3± 12.5

5.54± 0.21

18.5± 0.7

Table 2. Effects of initial peptone concentration on Plt production for the cultivation of P.M18R. a Cultivation time when the maximum Plt production was achieved. 3.1.3 Experiments on carbon/nitrogen ratio The combined effect of carbon (glycerol) and nitrogen (peptone) was studied using the CCD because the concentrations of both carbon and nitrogen sources and their ratio are very important for metabolite production (Chang et al., 2002; Li et al., 2008). The levels of variables for CCD experiments were selected according to the results of the one-at-a-time strategy, and the coded (−1.414, −1, 0, 1, and 1.414) and real values of the variables at various levels are listed in Table 3. The experimental responses, along with the predicted response obtained from the regression equation, are also shown in Table 3. Regression analysis was performed to fit the response function (Plt production) with the experimental data. From the variables obtained (Table 4), the model was expressed by Eq. (2), which represented Plt production (y) as a function of glycerol (x1) and peptone (x2) concentrations. Furthermore, the results of the F-test analysis of variance (ANOVA) in Table 5 showed that the regression was statistically significant (P < 0.05) at a 95 % confidence level. The model presented a high regression coefficient of 0.9694.

y(mg / L )  577.732  71.327 x1  54.053x2  46.802 x12  106.449 x22  24.010 x1 x2

(2)


76

x1 (g/L) Glycerol

Runs

y (mg/L) Plt production

x2 (g/L) Peptone

Observed

Predicted

1

21 (+1)

24 (+1)

444.8± 22.1a

2

21 (+1)

16 (-1)

593.9± 35.2

573.9

417.7

3

15 (-1)

24 (+1)

311.8± 17.5

323.1

4

15 (-1)

16 (-1)

364.9±20.4

383.2

5

22.242 (1.414)

20 (0)

553.4± 10.9

585.0

6

13.758 (-1.414)

20 (0)

405.9± 25.0

383.3

7

18 (0)

25.696 (1.414)

278.9±14.1

288.4

8

18 (0)

14.304 (-1.414)

441.8± 19.4

441.3

9b

18 (0)

20 (0)

548.1

577.7

10

18 (0)

20 (0)

573.6

577.7

11

18 (0)

20 (0)

602.1

577.7

12

18 (0)

20 (0)

589.6

577.7

13

18 (0)

20 (0)

575.1

577.7

Table 3. Experimental design and responses of the central composite design (CCD). a Samples were taken at 72h, and the standard deviation was calculated from three independent samples. b Runs 9-13 were replicates at the center point. Parameters

Parameter estimate

Standard error

T value

Pr > |t|

Intercept

577.732

11.746

49.184