STUDIES ON VARIABILITY IN Alternaria alternata ( KESSLER ) CAUSING LEAF BLIGHT OF ISABGOL ( Plantago ovata )

All the five isolates of Alternaria alternata isolated from different agro climate zone of Rajasthan were tested for their variability in terms of cultural, conidial, pathogenic characteristics and toxin production. All the five isolates differed in cultural characters i.e. dark black colored and very fast mycelial growth with smooth margins (90.00 mm), light black with white at centre and fast growing (80.00 mm), dark brown and medium mycelium growth with smooth margins (75.00 mm), black colored, medium flat mycelial growth with smooth margins (68.00 mm) and white with slightly black in colour with slow mycelial growth (65.00 mm) were observed in Aa-1, Aa-2, Aa-3, Aa-4 and Aa-5 respectively. The variability in conidial morphology of five different isolates was simple, septate, pale to dark brown in colour, often geniculate with one conidial scar. In respect of pathogenic variability, showed significant variations in terms of disease intensity and incubation periods. The isolates Aa-1 was highly pathogenic on Isabgol cv. RI-89 under artificial inoculation conditions showing 52.12% disease intensity followed by Aa3 ,Aa-2, Aa-4 and Aa-5 isolates. The variability in toxin production was reflected in terms of time taken in inducing wilting symptoms of Isabgol cuttings. Isolate Aa-1 was highly toxic followed by isolates Aa-2, Aa-3, Aa-4 and Aa-5.

Variability studies are important to document the changes occurring in populations and individuals as variability in morphological and physiological traits indicate the existence of different pathotypes.The variability is a well known phenomenon in genus Alternaria and may be noticed as changes in spore shape and size, growth and sporulation, pathogenicity, etc. Pathogen population structure and mechanisms by which variation arises within a population is of paramount importance for devising a successful disease management strategy.This requires continuous monitoring of the development of pathogen variability more so for the breeding programme aimed at developing resistant genotypes to the given set of pathogenic races (Sartorato, 2002).Variation in pathogen populations can generally be detected with methods like cultural, spore morphology, pathogenic and toxin variability.Hence, present investigations were carried out to study the variability in Alternaria alternata (Kessler) causing leaf blight of Isabgol (Plantago ovata).
Alteraria alternata (Fr.)Keissler.Pathogenicity test was done according Koch's postulates for all the five isolates.The identity of R.C.A. farm (Udaipur) isolate was confirmed by Indian Type Culture Collection (ITCC), Division of Plant Pathology, IARI, New Delhi-110012 (The ITCC Code no. 6317, 2008).The culture was purified by single spore technique.The single spore technique was conducted as per the method described by Sofi et al. (2013).

Cultural variability
For cultural variability, five isolates of the pathogen were grown on PDA to observe their growth pattern.The 5mm discs of pure culture of isolates were inoculated at the center of the pre poured Petri plates from 10 days old actively growing culture.All inoculated plates were incubated attemperatureof 25ºC±1ºC in BOD (Biological oxygen demand) incubator.Each isolate was replicated thrice.The growth rate was measured and colony characters, pigmentation, growth habit and sporulation were measured after 24 hrs of incubation till the growth of the pathogen in Petri plate completes.

Variability in spore morphology
To purify Alternaria culture, a conidial suspension was prepared in sterilized distilled water from 10 days old culture on PDA and flooded on 2% plain agar in Petri plates.The excess suspension was drained out and the Petri plates were then incubated in inverted position at 25 ±1ºC.After eight hours a single germinating spore was marked with the help of dummy objective and then transferred individually with a piece of plane agar medium to PDA slants by inoculating needle under aseptic conditions.These monoconidial isolates maintained on PDA slants were used to study spore morphology as described by Boedo et al. (2012).The observations on variation in conidial morphology of five isolates of A. alternata were recorded with the help of Ocular and Stage Micrometer.

Pathogenic variability
Pathogenic variability is the genetic character of fungi which may vary amongst isolates.Healthy seeds of Isabgol variety RI-89 were surface sterilized with 0.1% HgCl 2 and were sown @ 10 seeds per pot and replicated thrice.Leaves stem and branches of six weeks old plants were randomly selected, and these were injured gently by delicate brush and ten days old culture suspensions of isolates were sprayed with hand automizer in early morning hours, when dew deposition on leaves is there.Simultaneously, un-inoculated check was maintained by spraying sterilized distilled water on plants.The inoculated plants were observed daily to record the incubation period for the disease development.The disease intensity was calculated with the help of disease rating scale (1-5).The details of the rating scale are as follows.

Toxin variability
Several phytopathogenic species of Alternaria have been reported to produce phytotoxic metabolites, which play a significant role in pathogenesis and many of them have been chemically characterized (Devi et al., 2010a).For toxin variability, 25 mL Richards' medium (pH 6.5) in 100 ml sterilized flasks were inoculated with 5 mm diameter fungal bits of 10 days old culture of different isolates of Alternaria alternata grown on PDA and incubated at 25 ± 1ºC for 15 days.The culture filtrate was obtained by filtration through Whatman No.42 filter paper.The culture filtrates which were obtained from 15day old cultures of Alternaria alternata were centrifuged at 600 rpm for 20 min.The clear supernatant solutions were collected in clean sterilized conical flasks and pellet sedimented at the bottom of the centrifuge tube was discarded.The clear supernatant solutions served as samples of crude toxin produced by different isolates of Alternaria alternata used to study toxin variability, by using detached plant twigs dip method.Observations were recorded regarding toxicity symptom expressions like necrosis, leaf drooping, wrinkling and drying of leaves at regular intervals (6 hr, 12 hr 18 hr 20 hr and 24 hrs).

Cultural variability
All the five isolates showeddifferences in colony characters i.e. dark black colored and very fast mycelial growth with smooth margin., light black with white at centre and fast growing., dark brown and mycelial growth with smooth margin., black colored, flat mycelial growth with smooth margin, and white with slightly black in colored with slow mycelial growth were observed in Aa-1, Aa-2, Aa-3, Aa-4 and Aa-5 respectively.The average radial growth of isolate Aa-1 was highest i.e. 90.00 mm while, in isolate Aa-2, Aa-3, Aa-4 and Aa-5, it was comparatively less i.e. 80.00 mm, 75.00 mm, 68.00 mm and 65.00 mm respectively on 7 th day of incubation under uniform environments and medium.Sporulation was recorded in all five isolates but very good sporulation was observed in Aa-1.In view of the results obtained for cultural variation, it is clear that all the five isolates differed with respect to mycelial growth of Alternaria alternata attained after 7 th day for sporulation and colony characters (Table 1).
These results are in agreement with Pandey et al. (2005) where, they concluded that variability in Alternaria solani exists only with respect to cultural characters.Eleven isolates of A. solani causing early blight of tomato showed cultural variability in respect of radial growth, growth rate per day and pigmentation on potato dextrose agar.These results are also in similarity with the results obtained by Verma et al. (2007), Raja and Reddy (2007) and Tetarwal et al. (2008).
The present result was agreement with the results of Raja and Reddy (2007) whocollected the samples of leaf spot and fruit rot caused by Alternaria alternata from brinjal growing areas and it was found that the size of conidia varied from 35.2 -43.5 μm and 12.4-13.9μm wide, with average beak length of 9.6 -12.4 μm.Horizontal and vertical septations of conidia varied form 1.8 and 0.3, respectively and conidia were produced in chain.Similar results was also observed by Wagh et al. (2013) that seven days old culture of Alternaria alternata revealed hyaline, septate and branched mycelia, conidiophores with 30.0-80.2 μ length and 3-6 μ width and obclavate to obpyriform conidia (23-30 x 9.2-12.7 μ) with short conical beak arranged in acropetal fashion.

Toxin variability
The details of the experimental results are presented in Table 4.The culture filtrate is assumed as 100 per cent toxin concentration.This was simply an indicator test for toxin production.The symptoms like drooping of leaves, blackening of leaves was initiated at 6 hours and continued up to twentyfour hours, finally leading to wilting and necrosis, thus revealing the existence of variation among the isolates in producing toxic metabolites in the culture medium, which was reflected in terms of inducing wilting of Isabgol cuttings.The results indicated that Aa-1 isolate showed very severe toxic effect where initial toxicity symptom expression was within six hours, leading to complete and severe necrosis of leaves with distinct black colourations.Similarly, severely toxic , moderately toxic, slight toxic and least toxic effect were observed in cultural filtrate toxin of Aa-2, Aa-3, Aa-4 and Aa-5 isolates, respectively.This suggests that the toxin has active role in causing disease as well as mortality.
Such phytotoxic effects produced by culture filtrate were also reported by Reddy and Chaudhary (1990) where, they observed that, when pigeon pea seeds were soaked in culture filtrate of six Fusarium udum isolates for 6, 12, 24 h, no germination occurred after 24 h and radial length was also decreased with increasing in soaking time.Maiero et al. (1991) stated that Alternaria solani produced phytotoxic metabolites, and tomato seedlings exposed to culture filtrates for 20 h exhibited marginal and inter veinal leaf necrosis and subsequently wilting.
) 21-40% infection or 21-40% leaves of the plant are infected.(3)41-60% infection or 41-60% leaves of the plant.(4)61-80% infection or 61-80% leaves of the plant are infected.(5)81-100% infection or 81-100% leaves of the plant are infected.Numbers of plants in each score were recorded.Per cent disease index was calculated from these data following standard formula byWheeler (1969), as given: Where n = Number of plants in each score N= Total number of plant

Table 3 :
Pathogenic variability of five isolates Alternariaalternata under artificial inoculation conditions.
*Average of three replications; Figures in parentheses are angular transformed values
* Mean no. of 25 conidia and ± S.D. of mean value *

Table 1 :
Cultural variability among five isolates of Alternariaalternata on PDA

Table 4 :
Kumar et al. (2008)mong five isolates based on their culture filtrates (crude toxin) toxicity symptoms on Isabgol leavesThe pathogenic variability studies have also been carried out byVerma et al. (2007),Kumar et al. (2008)on A. solani.They recorded pathogenic variability among different isolates of A. solani.Tetarwal et al. (2008) observed variability among six isolates of A. alternata infecting Senna (Cassia angustifolia)