Pathogenicity and Morphological Variabilities of Fusarium oxysporum f.sp. cepae Isolates in Onion

Basal rot disease infected onion bulbs were collected from fifteen places of

Onion (Allium cepa var aggregatum G.Don) is one of the important vegetable crops grown in India. China ranks first in world onion production followed by India, USA, Turkey, Pakistan, Russia, Indonesia, Vietnam, and Myanmar. Basal rot is a devastating disease of onion caused by Fusarium oxysporum Schlechtend: Fr. f. sp. cepae (Hans.) (Coskuntuna and Ozer, 2008). In India the incidence of basal rot was first reported by Mathur and Shukla (1963). In Tamil Nadu, this disease was first observed by Ramakrishnan and Eswaramoorthy (1982) from Coimbatore district. The fungus attacks seedlings, causing pre and post emergence damping off, root rot of older plants, and stem plate discoloration and basal rot of bulbs in the field and in storage (Abawi and Lorbeer, 1972). It can significantly reduce the crop yield to approximately 45 t/ha in USA (Schwartz et al., 1991). In Japan, during summer more than 50 per cent loss occurs in welsh onion due to Fusarium basal rot (Dissanayake et al., 2009). Morphological characters are important tools in the identification and classification of fungus. Several workers described enormous pathogenic variability among Fusarium oxysporum f.sp. pisi isolates (Whitehead et al., 1992;Kraft, 1994). Singh et al., (2010) studied the variability in cultural characteristics and virulence among three isolates of Fusarium oxysporum f.sp. ciceri in chickpea under in vitro condition. Hence, the present investigation was conducted to study the morphological and pathogenic variability among isolates of Fusarium oxysporum f. sp. cepae.

Isolation of pathogen
Basal rot disease infected onion bulbs were collected from onion growing areas of Tamil Nadu, India during 2008-2009. The pathogen was isolated from the diseased tissues of onion by tissue segment method (Rangaswami, 1958). Infected portions of diseased plants were cut into small pieces using sterilized scalpel and surface sterilized with 0.1 per cent mercuric chloride for one minute and washed in three changes of sterile distilled water and then placed on Petri dish containing solidified Potato Dextrose Agar (PDA) medium. These plates were incubated at room temperature (28 ± 2ºC) for five days and observed for the growth of the fungus. The hyphal tips of fungi were transferred aseptically to PDA slants for maintenance of the culture.

Morphological variability among Fusarium oxysporum f. sp. cepae isolates
From the five day old culture plates nine mm culture disc of the pathogen was cut by a sterilized cork borer and placed at the center of each sterile Petri dish containing 20 ml of previously sterilized and solidified PDA medium. The plates were incubated at room temperature (28+2oC) for five days. The growth and morphological characters of the isolates viz., colony morphology, mycelial growth rate, colony colour, conidia size, shape and septation were observed, measurement was made under the microscope (Olympus BX41).

Pathogenic variability among Fusarium oxysporum f. sp. cepae isolates
The isolates of Fusarium oxysporum f. sp. cepae were multiplied on sand-maize medium (Riker and Riker, 1936). The medium containing sand and *Corresponding author email:malathi_agri@redifmail.com maize powder (19:1) was mixed, moistened with 400 ml of water kg-1 and then packed in polypropylene bags. The bags were sterilized in an autoclave at 1200C with 15 psi for 20 minutes and continued the sterilization for two concecutive days. Two nine mm PDA culture disc of actively growing Fusarium oxysporum f. sp. cepae were inoculated in each polypropylene bag replicated three times. They were incubated at room temperature (28±2ºC) for 15 days and used as source of inoculum Virulence of isolates of Fusarium oxysporum f. sp. cepae Earthen pots of uniform size (30 cm diameter) were filled with five kg of sterilized garden land soil. Five gram inoculum of each isolates (multiplied on sand maize medium) of the pathogen was mixed with soil present in pots. Four onion bulbs (cv. Co-5) were sown in each pot and replicated three times. The pots were maintained in green house by regular, uniform and careful watering and the growth was constantly observed for development of the disease symptoms. The per cent disease incidence of each isolate was recorded after 25 days of inoculation.

Results and Discussion
The mycelial growth habit of fifteen isolates was studied of which isolate DNSFOC1 significantly recorded 29.93 mm mycelial growth per day followed by ERVFOC2 which recorded 26.45 mm growth per day (Table 1). Minimum mycelia growth was observed in COAFOC3 (21.45 mm) isolate. The mycelial colours of isolates were light brown, dark brown, light yellow and pink. Compact, fluffy and sparse colony types were observed among the isolates. Fusarium oxysporum f. sp. ciceri isolates (Foc1, Foc2, Foc3) showed dull white, flat hairy growth and white fluffy colony with irregular margin (Singh et al., 2010) Fusarium oxysporum f. sp. cepae produced macroconidia and microconidia. The microconidia were oval to reniform while the macroconidia were falcate to straight, apical cell somewhat pointed. The septation of the macroconidia varied from 2-4 among the isolates. The length of macroconidia varied from 20.25 to 27.43 μm and the width ranged from 2.36 to 3.16 μm ( Table 1). The maximum conidial length and width were observed in DNSFOC1 (27.43 and 3.16 μm). The minimum conidial length was observed in THPFOC3 (20.25 μm). The minimum conidial width was observed in THBFOC2 (2.36 μm). Fusarium oxysporum f. sp. ciceri (foc2) showed 3 to 6 septa in conidia whereas Foc 1 and Foc 3 had 2 to 3 septate (Singh et al., 2010).
The length of microconidia varied among the isolates from 7.10 to 8.43 μm and the width ranged from 2.14 to 2.67 μm. Maximum conidial length and width was observed in DNSFOC1 (8.43 and 2.67 μm) and minimum conidial length was observed in ERKFOC1 (7.10 μm). Minimum conidial width was observed in DNTFOC3 (2.14 μm). Alice (1994) reported variation in morphological characters of Fusarium oxysporum in onion. Thirty isolates of Rhizoctonia bataticola collected from major chickpea growing areas were highly variable in their cultural and morphological characters (Manjunatha and Naik, 2011). The results indicated that difference in morphological character was positively correlated with its virulence.  Table 2). DNSFOC1 exhibited yellowing and wilting of leaves within twenty days after inoculation. The leaf tip dieback eventually progressed to the entire leaf and infected onion bulbs were pulled out easily.