Advances in Environmental Biology

Advances in Environmental Biology, 8(10) June 2014, Pages: 36-49

AENSI Journals

Advances in Environmental Biology ISSN:1995-0756 EISSN: 1998-1066

Journal home page: http://www.aensiweb.com/aeb.html

Isolation,

pathogenicity

test

and

oxysporum f.sp radicis lycopersici

physicochemical

studies

of

Fusarium

H.Benaouali, N. Hamini-Kadar, A. Bouras, S.L. Benichou, M. Kihal, J.-E. Henni. Laboratory of Applied Microbiology. University of Oran Es-Senia, Faculty of Science,Department of Biology, PO Box 1524,31000 Oran, Algeria. ARTICLE INFO Article history: Received 25 April 2014 Received in revised form 20 May 2014 Accepted 25 May 2014 Available online 22 June 2014

Key words: Fusarium oxysporum, pathogenicity test, various media, pH, temperature, light, humidity, Carbone, Nitrogen.

ABSTRACT The study of 27 isolates of F.oxysporum from different regions of western Algeria(Oran, Mostaganem, Chleff) Allowed on the basis of their macroscopic appearance to differentiate four morphotypes (downy, cottony, ras mucous and senescent) with different pigmentation (white, yellow, pink and purple).The pathogenicity test confirmed the presence of Fusarium oxysporum f.sp radicis lycopersici in the western region of Algeria. Of the 27 strains studied 22 have shown signs of crown and root rot, with very high degrees of virulence compared to the references strains and 5 isolates were not pathogen. For a better understanding of the ecology of these isolates a study of the influence of different physicochemical conditions were realized. The results showed a better growth of the isolates on PDA medium with an important pigmentation, mycelia growth of different isolates is quiet faster in the darkness than in light and the optimum pH for growth was 6 and 7 with a rate of humidity ranging from 74 to 80% and an optimal growth temperature of 23°C and 28°C.

© 2014 AENSI Publisher All rights reserved. To Cite This Article: H.Benaouali, N. Hamini-Kadar, A. Bouras, S.L. Benichou, M. Kihal, J.-E. Henni., Isolation, pathogenicity test and physicochemical studies of Fusarium oxysporum f.sp radicis lycopersici. Adv. Environ. Biol., 8(10), 36-49, 2014

INTRODUCTION The tomato (Lycopersiconesculentum Mill.) is one of the world’s most cultivated vegetable crops. Tomato plants are affected by several diseases, including the two formae speciales of Fusarium oxysporum : Fusarium oxysporum f.sp.lycopersici and Fusarium oxysporum f.sp. radicis-lycopersici [7]. F. oxysporum has spread worldwide and is phylogenetically diverse. The species is well known as a mycotoxin producer, and so its precise identification is of prime concern [22]. Fusarium oxysporum .f.sp.radicis-lycopersici (FORL) causes the disease crown and root rot of tomato (Jarvis and Shoemaker,1978).This disease has been reported in at least 32 countries [24]. FORL is one of the most destructive soilborne diseases of tomatoes occurring in greenhouse and field crops [40]. When tomato plants were affected by Forl, it appears first yellowing of older leaves and wilting of leaflets. Symptoms progress and plants wither and die together or it can persist in a state of weakness, infected plants may be stunted. FORL attack the roots grow within the conducting vessels of the roots and base of the stem, which turn brown and rot. Collar cankers are sometimes observed with fungus fructification facilitating the spread of the disease [32]. Fusarium oxysporum is is an anamorphic species identified by morphological criteria shared by both pathogenic and nonpathogenic strains [3]. Fusarium oxysporum produce sparse to abundant aerial mycelium with wite, pink, salmon and purple pigmentation on the reverse side of the colony in culture [29,18]. In study of variability in the morphology of Fusarium oxysporum f.sp.lycopersici have described 4 morphologic types fluffy, cottony, senescent and Slimy where pigmentation of thall and medium may vary within types: colorless - purple - pink - red - wine dregs on PDA . vinacious grey to purple and the under surface may be colorless or light purple pigment diffusing into agar [33]. Fusarium oxysporum produce three types of asexul spore: microconidia, macroconidia and Chlamydospores. Strains of F. oxysporum have been divided into formae speciales on the basis of virulence on a particular host or group of hosts [8]. Within F. oxysporum there is a high level of host specificity with over 120 described formae speciales capable of causing vascular wilt diseases of many agricultural crops. Further subdivisions of formae speciales into races are often made based on virulence to a particular set of differential host cultivars that vary in disease resistance [1]. The common characteristic of F. oxysporum strains is the Corresponding Author: H. Benaouali, Laboratory of Applied Microbiology. University of Oran Es-Senia, Faculty of Science, Department of Biology, PO Box 1524, 31000 Oran, Algeria.

37

H. Benaouali et al, 2014 Advances in Environmental Biology, 8(10) June 2014, Pages: 36-49

ability to parasitize plant roots, usually without inducing symptoms. This capability is non-specific and pathogenic strains can colonize roots of plants in which they will not cause disease [15]. The present work was realized For a clearer understanding of growth conditions of Forl by studying the effect of Carbon and Nitrogen sources, different media, temperature, pH ,humidity and light on mycelia growth of Forl after isolation ,identification and characterization of F.oxysporum from Tomato and confirmation of formae special and pathogenicity by greenhouse bio essay . MATERIALS AND METHODS Areas and sites surveyed : During the seasons from 2011 to 2012, 8 regions were visited between 2011 and 2012 (Table1). From each site, 10 plants showing symptoms of yellowing or wilting were collected for laboratory analysis.The infected plants were placed in paper envelopes, air-dried at room and stored at 18°C until used to isolate the pathogen. Table 1: Fusarium oxysporum isolates collected in this study Isolation Part Sampling site stem ITCMI Oran soil ITCMI Oran stem Boudjemaa Oran stem Hassibounif Oran stem Hassi ben okba Oran roots Hassi ben okba Oran soil Hassi ben okba Oran pivot StidiaMostaghanem collar StidiaMostaghanem stem OuledshailiaCHLEF collar CHLEF collar OuledshailiaCHLEF collar Hassibounif Oran root Sdimaarouf C3 Oran stem Sdimaarouf C1 Oran collar Sdimaarouf C2 Oran pivot Sdimaarouf C2 Oran pivot Sdimaarouf C3 Oran

Date 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012

Isolation of the pathogen from the infected plant and seed tomato: The isolation was carried out from the stems, the roots and the collar of the samples showing symptoms of the disease (Figure 1). The samples were washed with tap water to remove all visible soil; freshly infected tissue was selected for isolation. The infected tissue was cut into small pieces 5-10 mm long, disinfected with 2 % NaOCl for 3 min, then rinsed with distilled water and dried for 4 minutes on an absorbent water paper (sterilized). Small pieces were placed on a PDA medium (Potato Sucrose Agar 250 g of potato broth , 20 g agar and 20 g of sucrose per liter of distilled water). When the fungus was isolated directly from samples of soil, the soil is dried and sieved before sprinkle on PDA medium , or by dilution method , 1 g of soil was suspended in 9 ml of sterile distilled water, one gram of soil sample was suspended in 9 ml of distilled sterile water in test tube, this solution represents dilution 10-1, from this dilutions the dilution 10-4 were prepared and 0.5 ml were spread on the surface of PDA medium. All isolates were incubated at22°C for 5 days [37,27,35].

Fig. 1: FCRR symptoms observed in the samples. a)root system rots b,c )Longitudinal sections of the pivot of tomato plants infested by FORL, browning, d) collar rot, ,e )collar chancre.

38


Identification of isolates: The species Fusarium oxysporumis distinguished from other species of Fusarium in the production of microconidia, macroconidia and chlamydospores [5]. The isolates were subcultured on carnation leaf agar and observed to be producing macroconidia and microconidiophores was identified according to the keys of Fusarium oxysporum [5,29]. Only isolates with these characteristics are considered to belong to Fusarium oxysporumf Obtaining single-spore (clones): The spore cultures are obtained from isolates. Indeed, it has been shown [6] that the microconidia of Fusarium oxysorum and haploid uninucleate. The spore isolates are made as follows: a suspension of microconidia containing a suspension of 20 spores / ml is spread by streaking on PDA medium after 24 h of germination, the young fronds from one microconidie are aseptically collected and placed separately in Petri dishes. Seedling preparation: The tomato variety, Monfavet H63-5 F1, susceptible to Forl, Fol race 1, race 2, and race3 was used for this test. The seeds were first disinfected with sodium hypochlorite to 2% for 3min then rinsed in sterile distilled water three times and placed in a glass Petri dish containing filter paper soaked with sterile distilled water and incubated at 25 ° for 3 to 4 days until the appearance of the parenchyma. The sterile loam wasused for planting seeds.The seed Sowing was made at the rate of one seed per pot. The pots were placed into greenhouse illuminated between 20 and 22 ° C under daylight illumination. Watering was made daily, lightly and delicately with tap water. Preparation of the conidia suspension: FORL isolates to be tested was incubated at 28 ° C for 14 days on PDA medium. The spores were scraped from the surface of the culture and introduced into 10 ml of sterile distilled water. Concentration was determined by observation under an optical microscope using a Malassez cell. The suspension was then adjusted to the concentration of 106 spores / ml. Inoculation: Inoculation were performed at the four-true-leaf stage of plantlets (15 to 25days after sowing), the root system was cut to 7 mm below the pivot before being dipped in a microconidial suspension of the isolate to be tested. After 15 minutes inoculated plants were replanted to pots. The test was performed a rate of 10 replicates for each isolate, and ten plants were inoculated in distilled water as control, the reference strains were Fo47(nonpathogenic strain) Fol8 (pathogenic strain of Fusarium oxysporum f.splycopersici) and Forl12 (pathogenic strain of Fusarium oxysporum f.spradicis lycopersici) strain from the collection of microbiology laboratory of soil and environment INRA Dijon. After 3 weeks, they were uprooted and the lower stem and tap root were longitudinally sectioned for examination of internal tissues [25]. Symptom evaluation was made based on a rating scale of symptoms proposed by Vakalounakis and Fragkiadakis [39]. 0 = No symptoms; 1 = Light yellowing of leaves, light or moderate rot on taproot and secondary roots and crown rot; 2 = Moderate or severe yellowing of leaves with or without wilting, stunting, severe rot on taproot and secondary roots, crown rot with or without hypocotyls rot and vascular discoloration in the stem; 3 = Dead seedlings. Disease incidence percentage was determined using the following formula [38]: Diseas incidence % =

∑(scale × number of plants infected ) × 100 highest scale × total number of plants

Physicochemical study on the mycelia growth of Forl: In this study, all experiments were conducted in three replicas. Petri dishes were inoculated with 5 mm culture discs taken from the periphery of 10 days old cultures of Forl isolates With the exception of the study on the temperature, the cultures are incubated at 28 ° C. 7 days incubation, growth of the colony diameter was measured in two perpendicular axes. Effect of temperature: The effect of temperatureon growth was studied on PDA medium. The plates were incubated at 4 °C, 10 °C, 23°C, 28°C, 37°Cand 45 °C.

39


Effect of different culture media: Following four culture media were used to find out the most suitable one for the mycelia growth of the pathogen isolates :PDA, Czapeck, Malt, Mathur. Petri dishes containing the medium to study were inoculated with the isolates. Effect of different pH levels: The effect of pH on growth was studied on PDA medium; the pH medium was adjusted to 5,6, 7and 8 using either sterile 0.1 N NaOH or 0.1 N HCL. Effect of Nitrogen source: The Nitrogen source of the medium Czapek NaNO3 was substituted by organic Nitrogen source (Peptone, Arginine, and Asparagine) and by minerals Nitrogen source (NH4SO4, KNO3). Effect of carbon source: The Carbon source of the medium Czapek the Saccahrose was substituted by five sugars(glucose, dissacharides, lactose, Fructose, Maltose, and Starch),two alcools(Glycerol, Mannitol)and as acid(citric acid). Effect of light: The effect of light on growth was studied on PDA medium. The plates were incubated in the dark and other dishes were exposed to light and photoperid of 12 houres Effect of Salinity: The subcultured plates were turned and the covers were filled with 9ml of a saturated saline solution, the concentrations studied were, 14 %, 50 %, 74 %, 80 %, 95 %, and 100 %. Statistical Analysis of Data: Data of radial growth of Fusarium isolates were analyzed for standard deviation using Excel 2003. Results: Isolation and characterization:

Fig. 2: Different morphotypes obtained in this study.

40


A

B

D

C

E

Fig. 3: Microscopic observation of Fusarium oxysporum by the technique of CLA a:microconidia grouped false head, b:microindividual conidia, c:macroconidia d:subterminalspore, e:intercalary spore Table 2:Fusarium oxysporum isolates collected in this study,origin and macroscopic aspects Isolates Morphotype pigmentation isolation Part Sampling site F1 cottony whitish stem ITCMI Oran F2 cottony whitish soil ITCMI Oran F3 downy whitishgreen soil ITCMI Oran F4 downy purpel stem ITCMI Oran F5 downy whitishyellow stem Boudjemaa Oran F6 rasmucous whitish stem Hassibounif Oran F7 cottony whitish stem Hassi ben okba Oran F8 downy whitishpink roots Hassi ben okba Oran F9 cottony whitish soil Hassi ben okba Oran F10 rasmucous pink pivot StidiaMostaghanem F11 cottony whitish collar StidiaMostaghanem F12 downy whitishpink stem OuledshailiaChlef F13 downy whitish yellow collar Chlef F14 rasmucous whitishgreen collar OuledshailiaChlef F15 downy whitishpink collar CHLEF F16 downy whitishyellow collar Hassibounif Oran F17 cottony whitish collar Hassibounif Oran F18 cottony White collar Hassibounif Oran F19 downy White root Sdimaarouf C3 Oran F20 rasmucous purple stem Sdimaarouf C1 Oran F21 downy whitish collar Sdimaarouf C2 Oran F22 downy whitishyellow stem Sdimaarouf C1 Oran F23 cottony whitish pivot Sdimaarouf C2 Oran F24 downy whitish pivot Sdimaarouf C2 Oran F25 downy whitish pivot Sdimaarouf C2 Oran F26 cottony White stem Sdimaarouf C1 Oran F27 cottony whitish pivot Sdimaarouf C3 Oran

The pathogenicity test: results of the pathogenicity test are presented in table 3

Date 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012

41


Table 3:pathogenicity of Fusarium oxysporum isolated isolate Average of values Higher note observed in Number noted repetitions seedling F1 0.7 1 7 F2 0 0 0 F3 0 0 0 F4 0.9 1 9 F5 1.2 2 10 F6 0.8 1 8 F7 0 0 0 F8 1.8 2 10 F9 0.6 1 6 F10 0.7 1 7 F11 0 0 0 F12 1.2 2 10 F13 1.6 2 10 F14 0.9 1 9 F15 1.5 2 10 F16 1.8 2 8 F17 1 1 10 F18 1.6 2 10 F19 0 0 0 F20 1.3 2 10 F21 1.6 2 10 F22 1.7 2 10 F23 1.5 2 10 F24 1.7 2 10 F 25 1.4 2 10 F 26 1.7 2 10 F 27 1.6 2 10 Fo47 0 0 0 Fol8 2.6 3 10 Forl12 2.1 3 10 Distilled 0 0 0 water a FORL = Fusarium oxysporumf.spradicislycopersici, NON= Non-Pathogenic

Fol8

F8

of

infected

Fol8

Disease incidence% 23.33 0 0 30 40 26.66 0 60 20 23.33 0 40 53.33 30 50 60 33.33 53.33 0 43.33 53.33 56.66 50 56.66 46.66 56.66 53.33 0 86.66 70 0

F5

Fig. 4: inoculatedplants showed symptoms of yellowing and wilting of leaves and stunting

f.spa FORL NON NON FORL FORL FORL NON FORL FORL FORL NON FORL FORL FORL FORL FORL FORL FORL NON FORL FORL FORL FORL FORL FORL FORL FORL NON FOL FORL

42


Fig. 5: inoculated plants show rotting at the collar

Fig. 6: inoculated plants show rot at the pivot, roots and collar

e.d

Fo47

Forl12

F 21

F 22

Fig. 7: inoculated plants showed rot at pivot and second root.

F23

F 24

F 25

F26

43


Fig. 8: inoculated plants have no leaves or root symptoms. Physiochemical study: Effect of culture media: The results of the experiment of effect of culture media (Table 4) revealed that, PDA was the best medium for growth and sporulation of Forl among four medium chosen with a maximum growth of 82 mm and minimum growth of 61 mm. The grwoth on PDA medium was followed by Malt and Czapeck with a maximum growth of 80 mm and 77 mm, respectively and a minimum of growth of 60 mm and 40 mm respectively. For the Mathur Agar the isolates grew better than PDA but with a low sporulation. Table 4: Effect of different culture media on the mycelia growth of F.O.R.L: Isolat Colonydiameter (mm)* PDA Czapek F1 72±2** 60±1.73 F4 80±1 53±1.73 F5 80±0 50±0 F6 72±0.57 66±3 F8 72±2 52±1 F9 72±2 40±0 F 10 77±1.73 64±1 F 12 8±21.73 70±2 F 13 75±1.73 60±0 F 14 80±1.73 69±1 F 15 80±1 70±0 F 16 72±2 55±1 F 17 70±1 65±1.73 F 18 61±1.73 56±3 F 20 76±3 67±2 F 21 77±2 68±1 F 22 66±3 61±1 F 23 80±1.73 75±1.73 F 24 72±1.73 65±1.73 F 25 77±1.73 68±1 F 26 78±1.73 64±1 F 27 78±1.73 65±0 Forl12 80±1.73 72±2 *average of three replicates ** Value is the mean diameter ±SD

Malt 70±0 79±1 79±0 70±0 65±1 65±0 72±2 80±1 73±1 80±0 77±2 70±2 69±1 60±1.73 73±1.73 72±0 64±1 77±1 69±0 77±1.73 74±1 77±2 75±0

Mathur 82±0 82±0 82±1.73 82±0 74±2 78±1 78±1 82±0 78±1.73 82±0 82±1 75±1.73 73±1 64±1 80±1.73 82±1.73 68±1.73 82±1.73 76±3 78±1 78±1.73 79±1 82±0

Effect of Carbon sources: All the carbon sources were suitable for the fungus growth. However, for the sugars glucose was found to be the best carbon source followed by Lactose, Maltose and saccharose.for the alcoholics sources the majority of the isolate growth on Mannitol better than glycerol , on Citric acid the fungus grew but not as well as on sugars and Alcohols (Table 5).

44


Table 5: Effect of Carbon sources on the mycelia growth of. F.O.R.L Colony diameter (mm)* isolates monosaccharides disaccharides Polysaccharides Glu Fru Sac Malt F1 70±0** 75±0 60±1.73 72±2 F4 42±2 68±1 53±1.73 66±1.73 F5 57±2 57±0 50±1 47±0 F6 80±0.57 80±1.73 66±1.73 62±1.73 F8 82±0 73±1.73 52±1.73 76±3 F9 74±2 36±0 40±1 50±1 F 10 82±0 76±2 64±1.73 79±1.73 F 12 74±1.73 82±1.73 70±0 79±0 F 13 77±1 69±1 60±1.73 71±0 F 14 82±1.73 80±1.73 69±1 82±1.73 F 15 82±1.73 75±1.73 70±2 82±1.73 F 16 80±1.73 80±2.64 82±1 80±1.73 F 17 82±1.73 75±1.73 65±1 82±1.73 F 18 62±0 72±2 56±5.19 82±1.73 F 20 72±0 80±1.73 67±1.73 78±1.73 F 21 76±3 66±3.46 68±1 75±1.73 F 22 82±0 75±1.73 61±0 77±2 F 23 82±0 82±0 75±1.73 78±0 F 24 74±0 73±1 65±1 62±2.64 F 25 75±0 80±1.73 68±1 70±0 F 26 82±1.73 77±1 64±0 76±1.73 F 27 80±1.73 69±0 65±1 70±1 Forl12 82±0 72±0 72±2 82±0 *average of three replicates Glu=Glucose, Fru=Fructose, Sac= Saccharose, Malt=Maltose, AC=CitusAcide. ** Value is the mean diameter ±SD

Lac 58±1 65±1 61±1.73 76±1 82±1.15 53±1.73 80±1.73 82±1 80±1.73 80±1.73 82±1.73 82±1 82±1.73 80±1.73 78±1.73 82±0 65±1 61±1.73 76±1.73 82±0 82±0 53±1.73 80±1.73

alcohols

Ami 55±1 53±1.73 50±0 65±1 76±3 24±0 80±1.73 78±1.73 70±0 75±1.73 66±3 80±1.731 75±1.73 74±1 68±1 80±1.73 53±1.73 50±2 35±0 62±1.73 70±2.64 55±0 78±1

Cell 54±1.73 51±2 40±0 60±1.73 42±1 20±0 55±2.64 55±1.73 65±1 62±0 58±4.35 51±1 42±0 40±0 50±1 58±0 50±2 42±0 34±2.64 55±1.73 50±0 50±2 55±1

Gly 61±1 74±2 62±1 70±0 77±2 46±1.73 82±1.73 80±1.73 81±1 62±0 79±1.73 77±0 64±1 62±0 78±1.73 82±0 56±1.73 82±2 56±2.64 56±0 70±2 62±1 82±0

Man 63±1.73 77±2 67±1.73 74±0 80±1.73 50±0 72±1.73 82±1.73 72±0 60±1.73 82±1.73 80±1 67±0 67±2 74±2 74±2 50±2 70±0 63±1.73 60±1.73 50±4.16 60±1.73 72±2

Citric acid CA 44±1.73 63±1.73 48±0 52±1.73 57±0 53±1.73 58±0 65±1 58±1.52 52±2.30 61±1.73 57±2 53±1.73 48±1.73 60±1.73 60±1.73 48±0 60±4.35 44±1.73 44±0 50±0 44±2.64 58±2.64

Lac=Lactose, Ami=amidon, Cell=Cellulose, Gly=Glycerin, Man=Mannose,

Effect of nitrogen sources: Among the tree organic nitrogen sources tested, Peptone was found to be the best source of nitrogen for all the isolates of F. oxysporum . It was followed by Asparagine and Arginine. For the minerals nitrogen sources the best source was ammonium sulphate (NH4SO4 ) followed by Sodium nitrate (NaNO3 ) and Potassium nitrate (Kno3). (Table6) Table 6:Effect of Nitrogen sources on the mycelia growth of F.O.R.L isolates Colonydiameter (mm)* Organics Minerals Pep Asp Arg F1 80±1.73** 77±1.73 76±1 F4 79±1.73 77±2 76±0 F5 80±0 78±1.73 76±2.64 F6 81±2 78±0 75±1.73 F8 81±1 78±2 76±1.73 F9 83±1 80±1.73 79±1 F 10 79±2 76±2.64 72±1.15 F 12 79±1.73 70±0 68±1 F 13 80±1 77±2 70±0 F 14 82±1 78±1.73 77±2 F 15 81±2.64 80±1.73 75±0 F 16 75±0 67±1.73 60±1.73 F 17 80±1.73 78±1.73 77±2 F 18 82±0 80±0 76±1.73 F 20 80±1.73 79±0 76±0 F 21 82±1 77±0 70±2.64 F 22 81±2.64 77±2 76±1.73 F 23 82±1.73 77±2 75±0 F 24 80±1.73 70±1.73 65±1 F 25 82±1 78±0 75±0 F 26 80±1 78±1 75±0 F 27 82±1.73 79±1.73 77±1.73 Forl12 81±0 78±1.73 76±0 *average of three replicates Pep=Peptone, Asp= Asparagine, Arg=Argenine, ** Value is the mean diameter ±SD

KNO3 62±1 63±1.73 51±2 55±0 64±2.64 60±1.73 48±3.05 58±6.8 53±1.73 53±4.16 56±1.73 45±1 56±2 60±0 62±2.64 50±2.3 62±2.64 55±1.73 48±1.73 62±1 52±2 60±1.73 60±1.73

NaNO3 70±2 70±0 68±1 65±1 70±2.64 74±0 60±1.73 60±1.73 60±1.73 58±2.64 70±1.73 55±1.73 67±1 63±1 72±3.05 60±1.73 70±0 60±1.73 55±2.64 73±1.73 65±1 74±2 70±1.73

NH4SO4 75±1.73 75±0 75±1.73 74±2 75±1.73 78±1.73 68±1 64±1 64±1.73 75±1.73 72±1.73 58±2.64 75±0 74±1.73 74±2 64±2.64 75±0 73±0 58±2 75±0 74±0 76±1 75±0

45


Effect of temperature: The best temperatures were 23°C and 28°C .No growth was observed in any of the isolates evaluated at the temperature of 4°C and at 45°C. At temperature 28°C all the isolates showed very good growth with a maximum growth of 82 mm and the minimum of 68 mm .at 23°C the isolates grew well with 80 mm for the maximum growth and 61 mm for the minimum .At 10°C maximum growth of 52 mm and minimum of 35 mm .Low growth observed at 37°C with 30 mm as maximum and 18 mm as minimum growth. So 23 to 28 is the interval of a will growth of Forl (Table 7) Table 7:Effect of temperature on the mycelial growth of F.O.R.L isolates Colony diameter (mm)* 4°C 10°C 23°C F1 5±0** 46±2.64 75±2.645 F4 5±0 40±1.73 61±1.73 F5 5±0 44±2 70±1.73 F6 5±0 35±0 72±2 F8 5±0 46±1.73 75±0 F9 5±0 50±2.64 79±1.73 F 10 5±0 44±1 70±2.64 F 12 5±0 48±1 78±1 F 13 5±0 50±2.64 80±2.64 F 14 5±0 45±1.73 73±1.73 F 15 5±0 48±0 79±2 F 16 5±0 47±2.64 72±2 F 17 5±0 52±1.73 76±0 F 18 5±0 48±2 78±1.73 F 20 5±0 45±0 73±2.64 F 21 5±0 45±0 75±2 F 22 5±0 42±0 70±2.64 F 23 5±0 49±1 76±1.73 F 24 5±0 50±2.64 78±2 F 25 5±0 50±2 78±1.73 F 26 5±0 46±1 76±0 F 27 5±0 49±1.73 76±2.64 Forl12 5±0 50±0 80±1 *average of three replicates ** Value is the mean diameter ±SD

28°C 82±1 77±2.64 73±1.73 74±0 82±2 80±1.73 73±2.64 80±1 82±2.64 82±1.73 81±0 74±2.64 79±1.73 82±2 81±0 77±1.73 73±2.64 77±1 80±1.15 81±2 82±0 81±0 82±1

37°C 25±2 20±0 25±0 23±1.73 26±2.64 26±0 25±0 25±1.73 27±1.73 25±0 28±1 26±1.73 29±1.73 27±1.73 28±2 27±1 22±0 28±1.73 26±0 27±1.73 24±1.73 26±1.73 27±0

45°C 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0

Effect of relative humidity: In the study of the relative humidity , the maximum growth was observed in salinity 80%, 74 %, with a maximum diameter of 45 mm and 41mm respectively for the isolate F26.(Table8). Table 8: Effect of relative humidityon mycelialgrowth of F.O.R.L : isolat Colonydiameter (mm)* 14% 50% 74% F1 28±2.64** 35±3.78 37±2.64 F4 19±1.73 28±1.73 30±0 F5 25±2 33±2 35±2 F6 22±0 29±0 31±1.73 F8 30±0 32±1.73 34±2.64 F9 27±1.73 34±2.64 36±2.64 F 10 29±2.64 35±0 37±1.73 F 12 29±0 36±1 38±1.73 F 13 25±0 32±2.64 34±2.64 F 14 31±2.64 37±1 38±1.73 F 15 25±1.73 34±2.64 36±2 F 16 28±2.64 34±1.73 36±2 F 17 25±1.73 33±0 35±0 F 18 27±2 34±2 36±0 F 20 28±2.64 35±1.73 37±0 F 21 21±0 29±2.64 31±2 F 22 29±2.64 33±1.73 35±1.73 F 23 25±0 30±2 35±2 F 24 29±2.64 34±0 37±2.64 F 25 23±0 32±1.73 35±2.64 F 26 33±2.64 37±2.64 41±1 F 27 29±0 34±1 38±0 Forl12 28±1 35±1.73 37±2.64 *average of three replicates ** Value is the mean diameter ±SD

80% 40±0 34±2 37±1.73 34±1.73 36±1 40±2 40±0 41±2 37±0 42±0 40±2 39±1.73 37±2 39±1 40±2.64 36±0 40±0 38±0 42±2.64 38±1 45±2 42±2.64 40±0

95% 25±0 17±0 23±0 20±0 27±1.73 23±1 26±0 27±1.73 23±1.73 27±1 23±2 25±1.73 23±2 25±0 25±1.73 20±0 25±1 23±1.73 26±1.73 22±0 30±0 26±0 25±0

100% 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0 5±0

46


Effect of different pH levels: All the F.oxysporum isolates grew well at all the pH levels; Only F6, F10 and F25 who were different,they showed a best growth at pH 8 however maximum growth for almost of isolates was recorded at pH6 and pH7 (Table 9). The highest growth observed at pH6 was 82 mm for F4,F5, F21 and F.O.R.L 12. Least growth of all the isolates was observed at pH 5 the maximum growth in this level of pH was by F5 60mm. Table 9: Effect of different pH levels on the mycelia growth of F.O.R.L isolates Colonydiameter (mm)* pH 5 pH 6 F1 50±2** 76±1 F4 54±0.57 82±0 F5 60±2 82±0 F6 41±0 53±0 F8 54±1.73 79±0 F9 54±2.51 78±1.73 F 10 48±1 70±0 F 12 55±2.64 78±1.73 F 13 45±2 71±1.73 F 14 50±2 77±2 F 15 43±1.73 68±2.64 F 16 54±3.21 76±1 F 17 50±2 75±1.73 F 18 45±1 68±1 F 20 50±2 76±1.73 F 21 55±1.73 82±1 F 22 46±0 73±2.64 F 23 50±0 76±2.64 F 24 48±2 75±0 F 25 50±1 70±0 F 26 46±2.64 69±1.73 F 27 35±1.73 80±0 Forl12 52±2 82±0 *average of three replicates ** Value is the mean diameter ±SD

pH7 75±1.73 82±1.73 80±1.73 55±2.64 74±0 77±6.92 76±2.64 73±2 72±0 72±2.64 64±0 75±1.73 74±2 67±2.64 72±2.64 84±1 71±1 75±1.73 74±2.64 77±2 68±1 79±1.73 77±1

pH8 67±0 72±1 79±0 60±1.52 70±2.64 70±2 81±2.64 70±2.64 60±1 67±1.73 60±2.64 69±1.73 68±1 60±2.64 69±1.73 75±1.73 62±1.73 68±1 67±1.73 78±2 60±2 70±2.64 72±1.73

Effect of light: The growth of FORL was observed at darkness, light and 12 h photoperiod. F13 and F17 grew better under contnious light than 12h photoperiod and dark (Table 9). For the other isolates the maximum growth was observed at dark. There was a change in pigmentation for some isolates. Table 10: Effect of light on the mycelia growth of F.O.R.L isolat Colony diameter (mm)* light F1 78±1.73** F4 72±0 F5 67±1 F6 65±1 F8 74±1 F9 66±0 F 10 67±1 F 12 73±1 F 13 82±0 F 14 78±0 F 15 70±2 F 16 64±0 F 17 80±1.73 F 18 76±2.64 F 20 78±1.52 F 21 70±1.73 F 22 67±2.64 F 23 68±1 F 24 76±2 F 25 78±2 F 26 78±1 F 27 65±1 Forl12 73±1 *average of three replicates ** Value is the mean diameter ±SD

dark 81±1.73 75±1.73 70±2 73±1.73 81±1 77±2 72±0 76±1.73 76±0 82±1.73 78±1.73 70±2.64 70±0 80±1.73 81±0 78±1.73 77±2 78±1.73 80±1 82±1.73 81±1.73 78±0 82±0

12 hphotoperiod 80±1.73 74±2 69±0 70±0 80±1 75±1.73 69±1 73±0 80±1.73 80±0 74±2 69±1.73 75±1.73 77±2 80±1.73 73±1.73 69±0 75±1.73 76±1.73 80±1.73 80±1.73 75±1.73 80±1.73

47


Discussion: the four morphotypes described by [18] were observed in our collection: morphotype fluffy, cottony, senescent, Slimy (figure 2),(table 2).The most distinguishing characteristics of Fusarium oxysporum through the microscopic observation were the presence of falcate fusoid three to five septale macroconidia with pointed and curved ends , microconidia borne in false head on short monophialides. (Figure 3). The pathogenicity results showed the different pathogenic abilities of isolates .in the experiment only FOL 08 showed total death of the majority of 10 replicates seedling and the others showed stunting (figure 4 Fol 8).The reference strains FORL 12 were more pathogen then the isolates of our collection where the taproot and secondary roots of the majority of plants turned dark Brown plus the yellowing of leaves, the DI(Disease incidence) was 70%. The highest DI in our collection was 60% for the isolate F8 and F16. 13 isolate presente an DI ranged from 40 % and 56.66 % .Seven Isolates showed moderate pathogenicity, when the highest scale of 10 repetitions was 1, with maximum DI of 33.33 % for F17 and minimum of 20% for F9 .five isolates didn’t showed any symptoms (scale 0) and they were classified as non-pathogenic isolates (figure 8 )they were appeared like seedling inoculated in distilled water and F.O.47 fusarium oxysporum non pathogen. Osman et al., [30] studies the effect of various culture media on Fusarium oxysporum and PDA was the best culture media.These results were in confirmation with Ingole [20 who reported that PDA and Richard’s agar supported best mycelial growth of F. udum. In a study conducted by El-Sayed et al., [11]. PDA induced the best linear growth for F. oxysporum f. sp. lycopersici. In the present study our findings agree with that. PDA is one of the most commonly used culture media because of its simple formulation and its ability to support mycelial growth of a wide range of fungi. [36]. For the carbon sources we observed the same result of [21] in case of Fusarium oxysporum F. Sp. Ciceri when the glucose was the best carbon source followed by Maltose and it is similar to studies of [41] and [13] who found that glucose was the best carbon source for growth of Fusarium semitectum and Fusarium oxysporum f. sp. Ciceri respectively Peptone was found to be the best source of nitrogen for all the isolates of F. oxysporum. It was followed by Asparagine and Arginine and it was the best nitrogen source for Fusarium oxysporum F. Sp. Ciceri Farooqet al., [13] also Dandge, [9] showed that Asparagine was observed as an excellent Nitrogen source for growth of Fusarium oxysporum. Hibaret al., [19] showed that Fusarium oxysporum f.spradicis lycopersici grows well at temperature ranged from 20°C to 30°C. This results agree also with the result of [25] who found that the optimum temperature was at 26°C for all tested isolates Fol race1 Fol race 2 and Forl.and [14] where the Growth of F. oxysporumf.sp vanilla was maximum at 25°C, and [13] found that at 25°C and 30°C, Fusarium oxysporum f. sp. Ciceri attained the maximum growth and decline above 35 and drastically reduced below 15°C however no growth observed at 5° .similarly the temperature assay of [17] about Fusarium oxysporum f.sp. cubense revealed that the optimum temperature was 25°C for almost all isolates, At temperatures of 5 and 40◦C, no growth was observed for any of the isolates evaluated, while very little growth was observed at 10 and 35°C.And also 25 ± 2 °C was found to be most suitable temperature for mycelial growth of L. theobromaeand F. solani fungi [26]. The experiments of Popovski and Celar, [31] showed that the optimal growth occurred at 25 °C and 20-25°C for F. graminearum and F. culmorum respectively Gracia-Garza and Fravel [16] showed that Fusarium oxysporum f. sp. Erythroxyli produced more spores when exposed to fluctuating RH (100 to 75%). Baiyewu and Amusa, [2] investigated the effect of temperature and relative humidity on pawpaw fruit rot in South-Western Nigeria and shows that the relative humidity for maximum rot development of Fusarium moniliforme recorded ranged between 60-80% . [4] showed that F. oxysporum was found maximum radial growth at pH 6.5, followed by pH 7.5. Moussa [28] reported that Ph 7 gave the highest growth of Fusarium oxysporum f.sp lycopercisi. [14] found that the optimum pH for F. oxysporum f. sp. Vanilla isolates Was 5.0,Least growth of all the isolates was recorded at pH 9.0. [13] studied effect of pH levels on growth of Fusarium oxysporum F. Sp. Ciceri The fungus showed best growth at pH 7 and well growth observed at pH 6 [21] showed optimum pH for growth of F. oxysporumf.sp. ciceri ranged from 6.5 to 7.0. The maximum growth of FORL was observed at dark,Similar observations were made by [34] in the case of Fusarium solani .there was a change in pigmentation for some isolates .It was reported that darkness favors maximum uredospore germination of legume rust pathogen Uromycesviciae-fabae [12]. Conclusion: Pathogenic tests showed that all pathogenic isolates of Fusarium belongs to Fusarium oxysporum f.sp radicis lycopersici. Physiological studies revealed in this study that Fusarium oxysporum f.sp radicis lycipercisi showed a maximum growth on PDA medium at 23-28°C, pH 6 with 80% of relative humidity and darkness. Glucose was found to be the best source of carbon, where peptone was the best source of nitrogen. These

48


findings create bases for the culture and characterization of Fusarium oxysporum f.sp radicis lycopercisi for better understanding of their ecology. REFERENCES [1] Armstrong, GM., J.K. Armstrong , 1981. Formae speciales and races of Fusarium oxysporum causing wilt diseases. In: Fusarium Diseases, Biology and Taxonomy. NelsonPE, Tousson TA, Cook R.J. Ed. Pennsylvania State University Press, University Park. pp: 391-399. [2] Baiyewu, R.A and N.A .Amusa, 2005.The Effect of Temperature and Relative Humidity on Pawpaw Fruit Rot in South-Western Nigeria. World Journal of Agricultural Sciences, 1(1): 80-83. [3] Bao, J.R., D.R Fravel., N.R O’Neill., G. Lazarovits and P.V. Berkam, 2002. Genetic analysis of pathogenic and nonpathogenic Fusarium oxysporum from tomato plants. Can. J. Bot. 80: 271-279. [4] Bhale, U.N., 2012. Physiological studies of fungicide resistant and sensitive Fusarium oxysporum f.sp. Spinaciae. International Journal Of Ayurvedic And Herbal Medicine. 2:1 171:175. [5] Booth, C.,1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, pp: 237. [6] Buxton, E.W., 1954. Heterokaryosis and variability in Fusarium. oxysporum.f. sp. gladioli J Gem, Micro, 10: 71-8. [7] Çolak, A and M. Biçici, 2013. PCR detection of Fusarium oxysporum f. sp. radicis lycopersici and races of F. oxysporum f. sp. lycopersici. Turk J Agric For., 37: 457-467. [8] Correll, J.C.,1991. The relationship between formespeciales, races, and vegetative compatibility groups in Fusarium oxysporum. Phytopathology, 81: 1061-1064. [9] Dandge,V.S., Effect of nitrogen sources on the growth of different species of Curvularia, Fusarium, Phoma and Botryodiplodia.Journal of Experimental Sciences, 3(3): 24-27 [10] ELANi, A.S., 1968. The cytogenetics of the conidium in Microsporum gypseum and of pleomorphism and the dual phenomenon in fungi. Mycologia, 60: 999-1015. [11] El-Sayed, A. Fayzalla., Yasser M. Shabana and Nasser S. Mahmoud, 2008. Effect of Environmental Conditions on Wilting and Root Rot Fungi Pathogenic to Solanaceous Plants. Plant Pathology Journal., 7: 27-33. [12] Emeran, A.A., J.G. Sillero, R.E. Nikas and D. Rubiales, 2005. Infection structure of host specialized isolates of Uromycesviciae-fabae& of other species of Uromycesinfecting leguminous crop. Pl.Dis., 17: 17. [13] Farooq, S., S. M Iqbal and C.A. Rauf, 2005. Physiological studies of Fusarium oxysporum f. sp. ciceri. Int. J. Agri. Biol.,7(2): 275-277. [14] Gangadhara, N.B., R. Nagaraja, M .K. Basavaraja and N.R. Krishna, 2010 .Variability studies of Fusarium oxysporum f. sp. Vanilla isolates. International Journal of Science and Nature. 1(1): 12-16. [15] Gordon, T.R and R.D. Martyn, 1997. The evolutionary biology of Fusarium oxysporum. Annual Review of Phytopathology, 35: 111-128. [16] Gracia-Garza, J.A and D.R. Fravel, 1998. Effect of Relative Humidity on Sporulation of Fusarium oxysporum in Various Formulations and Effect of Water on Spore Movement Through Soil. Phytopathology, 88(6): 544-9. [17] Groenewald, S., N .Van den Berg, W.F.O. Marasas and A.Viljoen, 2006. Biological, physiological and pathogenic variation in a genetically homogenous population of Fusarium oxysporumf.sp. cubense. Australasian Plant Pathology, 35: 401-409. [18] Henni, J.E., C. Boisson and J.P. Geiger, 1994. Variability in the morphology of fusarium oxysporumf.sp.lycopersici. Phytopath. Medit. 33: 51-58 (Original Article in French). [19] Hibar, K., M. Daami-Remadi, H. Jabnoun-khiareddine and M. El-Mahjoub, 2006. Temperature effect on mycelial growth and disease incidence of Fusarium oxysporumf.spradicislycopersici .Plant pathology journal, 5(2): 233-238. [20] Ingole, M.N., 1995. Estimation of losses, variability among isolates and management of pigeonpea wilt caused by Fusarium udum Butler MSc (Ag.) Thesis, Dr. PDKV, Akola, pp.146.In Gangadhara N B, Nagaraja R, Basavaraja M K and Krishna N R (2010) .Variability studies of Fusarium oxysporum f. sp. vanillaeisolates. International Journal of Science and Nature., 1(1): 12-16. [21] Imran Khan, H.S., M. Saifulla, S.B. Mahesh and M.S. Pallavi, 2011. Effect of different media and environmental conditions on the growth of Fusarium oxysporum f. sp. Ciceri causing fusarium wilt of chickpea. International Journal of Science and Nature. 2(2): 402-404. [22] Irzykowska, L., J. Bocianowski, A. Waskiewicz, Z. Weber, P. Golinski, Z. Karolewski, M. Kostecki and W Irzykowski, 2012 .Genetic variation of Fusarium oxysporumisolates forming fumonisin B1 and moniliformin. J Appl Gen., 53: 237-247. [23] Jarvis,W.R and R.A. Shoemaker, 1978. Toxonomic status of Fusarium oxysporum causing foot and root rot of tomato.Phytopathology, 68: 1679-1680.

49


[24] Jones, J.B., J.P. Jones, R.E. Stall and T.A. Zitter, 1991. Compendium of Tomato Diseases. American Phytopathological Society, St. aul, MN, U.S.A. [25] Kim, J.T., I.H. Park, Y.L. Hahm and S.H. Yu, 2001. Crown and Root Rot of Greenhouse Tomato Caused by Fusarium oxysporum f. sp. radicis-lycopersici in Korea.Plant Pathol. J. 17(5): 290-294. [26] Kausar, P., S. Chohan and R. Parveen, 2009. Physiological studies on Lasiodiplodiatheobromae and Fusarium solani, the cause of Shesham decline.Mycopath.7(1): 35-38. [27] Messiaen, C.M et R. Cassini, 1968. Recherche sur les fusarioses. IV- La systématique Fusarium. Ann. Epiphyt. 19: 387-454. [28] Moussa MMA, 2004. Biological and biochemical aspects of Fusarium wilt diseases. PhD thesis, Fac. Sci. Damietta, Mansoura University. Egypt. [29] Nelson, P.E., T.A.Toussoun, W.F.O. Marass, 1983. Fusarium species: An illustrated manual for identification. The Pennsylvania State University Press, University Park. p: 193. [30] Osman, M., M.A. El Sayed, Y.A.H .Mohamed., M. Metwally, 1992. Effect of various culture conditions on alternaria alternata and fusarium oxysporum 1 culture media temperature age and carbon source. Microbios.,71(286): 15-26. [31] Popovski, S and F.A. Celar, 2013. The impact of environmental factors on the infection of cereals with Fusarium species and mycotoxin production – a review.ActaagriculturaeSlovenica. 101 str. 105-116. [32] Paulus, A.O., 1991. Fusarium Crown and Root Rot, pg. 14.In Jones JB., Jones JP, Stall RE and TA Zitter (eds.), ompendium of tomato diseases.American Phytopathological Society Press, St. Paul, MN. [33] Rozlianah, F.S. and M. Sariah, 2006. Caracterization of Malaysian Isolates of Fusarium from Tomato and pathogenicity testing.Research .J. of Microbiology, 1(3): 266-272. [34] Ramteke, P.K and S.S. Kambel, 2011. Physiological studies in Fusarium solani causing rhizome rot of ginger (Zingiberofficinalerosc.).The bioscan an inter.quaterly .J.of life sciences, 6(2): 195-197. [35] Rapilly, F., 1968. Les techniques de mycologie en pathologie végétale. Ann. Epiphyties, 19(no hors série), 102p. [36] Sharma, G. and R.R. Pandey, 2010. Influence of culture media on growth, colony character and sporulation of fungi isolated from decaying vegetable wastes. Journal of Yeast and Fungal Research, 1(8): 157-164. [37] Snyder, W.C and H.N. Hansen, 1954. The species concept in Fusarium with reference to discolor and other section. Am. J. Bot. 27: 738-742. [38] Song, W., L. Zhou, C. Yang, X. Cao, L. Zhang and X. Liu, 2004. Tomato Fusarium wilt and its chemical control strategies in a hydroponic system. Crop Prot., 23: 243-247. [39] Vakalounakis, D.J. and G.A. Fragkiadakis, 1999. Genetic diversity of Fusarium oxysporum isolates from cucumber: Differentiation by pathogenicity, vegetative compatibility and RAPD fingerprinting. Phytopathology. 89: 161-168. [40] Wojciech, S., S. Mirosława and H. Hanna, 2013. Fusarium oxysporum f.sp. radicis lycopersici – the cause of fusarium crown and root rot in tomato cultivation .Journal of plant protection research, 53: 2. [41] Yonggang Li, 2011. Biology Characteristic Determination of Fusarium Semitectumin Soybeans. International Conference on Agricultural and Natural Resources Engineering Advances in Biomedical Engineering, 3-5.