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acter species associated with citrus huanglongbing. Journal of Microbiological Methods 66: 104-115. Li R., Mock R., Huang Q., Abad J., Hartung J., Kinard G.,.

Journal of Plant Pathology (2014), 96 (3), 497-506

Edizioni ETS Pisa, 2014

Hartung et al.

497

LACK OF EVIDENCE FOR TRANSMISSION OF XYLELLA FASTIDIOSA FROM INFECTED SWEET ORANGE SEED J.S. Hartung1, S. Nian2, S. Lopes3, A.J. Ayres3 and R. Brlansky4 2Department

1USDA ARS Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD, USA of Pathogen Biology, School of Basic Medical Sciences, Luzhou Medical College, Sichuan, People’s Republic of China 3Fundecitrus, Araraquara, São Paulo, Brazil 4Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA

SUMMARY

INTRODUCTION

Citrus variegated chlorosis is among the principle diseases that affect sweet orange in Brazil and Argentina, and is viewed as an emerging threat by the U.S. sweet orange industry. The disease is caused by the fastidious bacterium Xylella fastidiosa. We have tested seed extracted from fruit heavily infected and symptomatic for X. fastidiosa infection as well as seed obtained from normal asymptomatic fruit from the same trees in Brazil. Seeds from symptomatic fruit were tested by PCR and found to be contaminated with X. fastidosa, and to weigh less than seeds from normal-sized fruit. Seed from both symptomatic and asymptomatic fruit were sown in the greenhouse and studied following germination. The growth of shoots, but not of roots, was reduced in seedlings produced from seed collected from symptomatic as compared to asymptomatic fruits. Symptoms of citrus variegated chlorosis were not observed in any of the seedlings derived from seed from symptomatic and asymptomatic fruit. Roots and leaf midribs from 260 seedlings were tested for the presence of X.  fastidiosa by PCR three months after sowing. No positive PCR results were observed. Leaf midribs from 148 seedlings were tested subsequently at 17 months after sowing using both standard format and qPCR assays, also without any positive results. Leaf midribs from 349 seedlings from a second seed harvest were tested seven months after sowing by qPCR also without any positive tests. Thus in this study no evidence for the vertical transmission of X. fastidiosa through contaminated seed was obtained.

Citrus variegated chlorosis (CVC) is one of many diseases of trees and tree fruits caused by strains of Xylella fastidiosa (Chang et al., 1993; Hartung et al., 1994). The pathogen and the diseases caused by it are very widespread in the Americas (Janse and Obradovic, 2010; Purcell, 1997; Purcell and Hopkins, 1996) but a recent outbreak was reported from southern Italy (Saponari et al., 2013; Cariddi et al., 2014). Other strains of the pathogen cause well known diseases including Pierce’s disease of grapevine, almond leaf scorch, phony peach and numerous diseases of shade trees usually observed in urban environments. X. fastidiosa is a member of the gamma proteobacteria (Wells et al., 1987) and is transmitted by a number of sharpshooter leafhoppers that feed in the xylem vessels which in turn are colonized by X. fastidiosa. There are four subspecies of X. fastidiosa: subsp. fastidiosa (Pierce’s disease), subsp. multiplex (affecting many fruit and shade trees in North America), subsp. pauca (citrus and coffee in South America, primarily olive in Italy) and subsp. sandyi (oleander). Members of the subspecies have been grouped by DNA sequence comparisons of the 16S RNA and the 16S-23S intergenic region (Chen et al., 2000) as well as by bulk hybridization of the total DNA of strains (Schaad et al., 2004). X. fastidiosa strains grow slowly in culture, require specialized media and are difficult to isolate (Davis et al., 1978). For this reason a number of PCR-based assays for the detection of the pathogen have been developed in both standard and quantitative formats (Li et al., 2013; Minsavage et al., 1994; Pooler and Hartung, 1995a). The citrus strain of X. fastidiosa was the first plant pathogenic bacterium to have its genome fully sequenced (Simpson et al., 2000), an indication of the importance of the disease that it causes. The genome of the Pierce’s disease ‘Temecula’ strain and two almond strains have also been sequenced and compared with that of the citrus strain (Van Sluys et al., 2003; Chen, 2010). CVC is a major production problem for sweet orange in Brazil, and eastern Argentina. In Brazil, the disease is spread throughout the country and is especially severe in the hot climate production regions of central and northern São Paulo state. A survey in 2010 estimated an infection

Key words: Xylella fastidiosa, citrus variegated chlorosis, qPCR, sweet orange

Corresponding author: J.S. Hartung Fax: +1.301.5045449 E-mail: [email protected]

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Xylella fastidiosa and sweet orange seed

Journal of Plant Pathology (2014), 96 (3), 497-506

Fig. 1. A. Foliar symptoms of citrus variegated chlorosis in sweet orange cv. Pera in the grove where fruit was harvested for this study. B. Undersized fruit harvested to extract seed for this study. A normal-sized fruit is at the upper right.

Journal of Plant Pathology (2014), 96 (3), 497-506

rate of sweet orange trees in Sao Paulo of 35%. Yield losses to CVC can be as high as 90%. Symptoms include a bright asymmetrical chlorosis with necrotic spots visible on the lower surface of the leaves (Hartung et al., 1994). The chlorosis may resemble zinc deficiency (Chagas et al., 1992; Rossetti et al., 1990) and zinc is accumulated by X. fastidiosa when it forms biofilms (Cobine et al., 2013). Other symptoms include stunting, defoliation and the production of characteristic small and hard fruit that lack juice. X. fastidiosa colonizes the sweet orange tree systemically. It can easily be found in the leaf midribs (Chang et al., 1993), in the fruit peduncle and in fruit (Rossetti et al., 1990) and can be transmitted by root grafting (He et al., 2000). This prompted an investigation of the possibility that the pathogen may be vertically transmitted through infested seed, and indeed evidence was found that suggested that X. fastidiosa could be transmitted through sweet orange seed (Li et al., 2003). In this study seed was collected from very small hard and symptomatic fruits. However, other apparently normal fruits are also found on the same trees. This study was undertaken to confirm that X. fastidiosa could be transmitted through seed and further to determine if such transmission could occur through seed collected from non symptomatic fruits. To do this, methods similar to the previous study were used with the exception that improved quantitative PCR-based assays specific for the pathogen at both the genus and subspecies level were added (Li et al., 2013), and it was possible to maintain the seedling trees for a longer period in the greenhouse to allow for repeated sampling as the seedlings increased in size. MATERIALS AND METHODS

Collection of fruits from CVC-affected trees. An orchard was selected located some 20 Km from Araraquara, an area with a high incidence of CVC. Trees of cv. Pera sweet orange grafted on Rangpur lime, 6 years of age, showing only CVC symptoms were selected for this study, though co-infection with Huanglongbing (HLB) was not ruled out. Typical symptoms of CVC were observed, including asymetrical chlorosis with necrotic spots on the leaves, dieback, and many fruits with a greatly reduced size (Fig. 1). The trees in this grove had previously been tested by PCR to confirm infection with X. fastidiosa subsp. pauca. From 10 selected trees fruits were collected to fill two plastic baskets of 60 liters volume each, one with fruits from symptomatic branches and one with fruits from asymptomatic branches from the same affected trees. In 2010, to fill the basket 379 fruits from the symptomatic and 173 fruits from the asymptomatic branches were needed; this observation is a reflection of the smaller size of the CVC symptomatic fruit. The symptomatic and asymptomatic fruits yielded 216 and 133 g of partially dried seeds, respectively. Seeds were washed with water then dried for one day prior

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to being shipped by express mail, under permit, to the USDA, ARS in Beltsville (Maryland, USA). Some seed developed superficial fungal growth. Dissection and inspection of the seed revealed that the fungus was a superficial infestation only, so it was not considered to be a problem for germination. In 2011 seed was collected following the same protocol. Seeds from symptomatic branches weighed 236 g, whereas those collected from asymptomatic branches weighed 140 g. Sowing of seeds in the greenhouse. In 2010, seed from healthy and CVC symptomatic fruits were received on August 23rd and sorted into six aliquots of 50 seeds each. Each of the 12 aliquots was weighed and data recorded. Then the 12 aliquots of seeds were taken to the greenhouse and sown into market pack flats filled with moistened Metro Mix 510 potting mix. Seeds were sown into furrows ca. two cm deep. For each set of six aliquots, three seed aliquots were sown directly; the other three had a small incision with a budding knife to allow a small peel of the seed coat to be pulled back prior to sowing to speed germination (scarification). The number of germinated seedlings was counted weekly to determine if the seed scarification treatment promoted germination. These data were evaluated with two-tailed t-tests. The greenhouse was maintained at 65-80 F (18-27°C) for most of the year, but the temperature reached 95 F (35°C) in the summer months. Ambient light was supplemented with high pressure sodium vapor lighting on cloudy days and throughout the winter season to extend the photoperiod. Flats were watered as needed with water containing nitrogen/phosphorus/potassium (100/25/100 ppm), copper (2 ppm) and iron (6 ppm). In 2011 seeds were received on July 26th collected from fruits as described for 2010 and were sown into flats of Metromix as described above. Three months after sowing seedlings were randomly assigned to thirteen groups of 20 designated by letters A-M. Soil was shaken off roots and each group of 20 seedlings was photographed. The roots and shoots were then measured to record the greatest length, the plants were divided and the roots and shoots were weighed separately. These data were evaluated with two-tailed t-tests. Two cm of the main root were cut out used to extract DNA with the DNeasy plant mini kit (Qiagen, USA). The midribs from two leaves from each seedling were also removed and used to extract DNA with the Qiagen-based protocol. The remaining 493 seedlings grown from seeds collected from symptomatic fruits and 150 seedlings grown from seeds collected from asymptomatic fruits were transplanted into 4×21 cm containers. One half of the seedlings received water only and the other half received the fertigation mixture above. This was done to induce nutrient stress in one set of plants to see if that treatment would enhance symptom expression (Cobine et al., 2013; Fuente et al., 2013). Throughout the experiments seedlings

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were sprayed with pesticides if needed. Soil was drenched with fungicides Banrot or Truban to control root rots. DNA extraction and PCR tests. DNA was extracted from seed coats, seed embryos, leaf midribs and root segments using the DNeasy protocol as previously described (Li et al., 2008). Approximately 100 mg of tissue were cut with a fresh razor blade into small pieces, then put into sealed plastic tubes and pulverized in a bead mill (Fast Prep, MP Biomedicals, USA) with steel BBs and Qiagen solution. DNA was quantified with a Nanodrop spectrophotometer (Thermo Scientific, USA). The DNA was tested by several sets of PCR primers specific for X. fastidiosa in both standard and TaqMan based qPCR formats (Table 1). This was done so that the results of one set of PCR primers could be confirmed with an independent PCR assay. The primer sets used included well known primer pairs 272-1 int and 272-2 int (Pooler and Hartung, 1995b) and RST (Minsavage et al., 1994). In addition, another primer pair was developed based on the genome sequence data of X. fastidiosa strain 9a5c (Simpson et al., 2000). This primer pair, designated XF2084 amplified DNA encoding a component of a multi drug efflux system found in citrus strains of X. fastidiosa (H. Hasegawa and J.S. Hartung, unpublished). The sequences of this primer pair are XF2084F 5’-TTCAAGGCGCATTCGCCACA-3’ and XF2084R 5’-AAGGTCGCTTTGAACCCGG-3’. Table 1. PCR primers and assays used in this study. Primers

Format

Target

Reference

272-1 int & 272-2 int

Standard

RAPD PCR Product

Pooler and Hartung, 1995

RST 31/33

Standard

16S rDNA

Minsavage et al., 1994

XF2084

Standard

Multidrug Efflux Protein

XF16S fpr

qPCR

16SrDNA

Li et al., 2013

CVC fpr

qPCR

O antigen acetylase

Li et al., 2013

Table 2. Weight of samples of 50 g of sweet orange seed obtained in 2010 from normal and small fruits affect with CVC. Sample

Asymptomatic Fruit

Symptomatic Fruit

1

5.69

5.24

2

5.87

4.79

3

5.97

5.24

4

5.23

4.75

5

5.58

5.41

6

5.82

5.08

Total

34.16

Mean



Journal of Plant Pathology (2014), 96 (3), 497-506

Xylella fastidiosa and sweet orange seed

5.69 ±

30.51 0.12a

5.08 ± 0.12b

a, b The means followed by different letters are significantly different. Two-tailed t = 3.17 (P 

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