Chitosan and yeast elicitor in suppressing seed-borne fungi of cucurbitaceous vegetables

ARTICLE INFO Experiments were conducted under laboratory condition to examine the efficacy of Chitosan and Yeast Elicitor to suppress the growth of seed-borne fungi of cucurbitaceous vegetables. Seeds of bottle gourd, sweet gourd, snake gourd, wax gourd and cucumber were collected from seed traders of Mymensingh districts and different seed borne fungi were isolated, purified and identified. Fourteen fungal species belonging to twelve genera consisting of Aspergillus flavus, Aspergillus niger, Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phoma exigua, Rhizopus stolonifer, Macrophomina phaseolina, Penicillium spp., Curvularia lunata, Chaetomium spp., Colletotrichum spp., Cercospora spp. and Alternaria alternata were isolated and identified. Four concentrations of Chitosan and Yeast Elicitors solutions (200, 500, 1000 & 2000 ppm) including one positive control Vitavax-200 WP (0.35%) were evaluated for controlling seed-borne fungi. Among the seed treating agents Chitosan (2000 ppm) and Yeast Elicitor (2000 ppm) showed better performance in suppressing the seed-borne fungi. Chitosan (2000 ppm) showed superior performance than Yeast Elicitor (2000 ppm). Results from the present study revealed that application of elicitors as seed treatment is a potential alternative of chemical fungicide for selective vegetables. Article history: Received: 26 February 2018 Accepted: 07 July 2018


Introduction
Cucurbits are important vegetable crops, not only in Bangladesh, but also in many other countries all over the world.Cucurbits belong to Cucurbitaceae family, which include bottle gourd (Lagenarea siceraria L), sweet gourd (Cucurbita moschata), snake gourd (Trichosanthes cucumeria), wax gourd (Benincasa hispida) and cucumber (Cucumis sativus) etc. Cucurbits are commonly exposed to attack by many serious soil-borne and seed-borne pathogens.Pathogen free healthy seeds are essential for desired plant populations and a good harvest.Of the 16% annual crop losses due to plant diseases, at least 10% loss occurs due to seed-borne diseases (Fakir, 1983).Coincidentally important or devastating crop diseases are seed-borne and caused by fungi.In addition, seed borne fungi are responsible for poor quality seeds in many crops (Neergaard, 1979).
For suppressing seed-borne fungi various elicitors can be used.Yeast Elicitor and Chitosan (β-1,4 linked Dglucosamine) are two important bio-polymers, can be commercially derived from various crustaceans commonly from the exoskeleton of shrimps and crabs (Boonlertnirun et al., 2008).These two products can modulate various cellular function including reactive oxygen production, ion channel activity through phosphorylation and dephosphorylation of target protein, stomatal movement, upregulation of pathogenesis related genes (Khokon et al. 2010).Both Yeast Elicitor and Chitosan can be used as seed treating agents and foliar application of these components can induce resistance to overcome the seedling diseases as well as final crop production (Mondal et al., 2013).Properties of chitosan for inhibition of pathogenic bacteria and fungi in antimicrobial films and edible coatings are used.Antimicrobial activity of chitosan resulting from positively charged amino groups.This group responds to negatively charged cell membranes of microorganisms.This reaction leads to the leakage of intracellular protein components and other microorganism components (Yarahmadi et al. 2014).Yeast extract (YE) is an important elicitor and is found to be rich in vitamin Bcomplex.It also contains essential components like chitin, N-acetyl-glucosamine oligomers, glucan, glycopeptides and ergosterol (Boller, 1995); these compounds elicit plant defense responses by triggering metabolite synthesis (Putalun et al., 2007;Cai et al., 2012).Yeast Extract has successfully been used in culture and overproduction of important phytocompounds were observed in several studied of plant genera (Prakash and Srivastava, 2008;Zhao et al., 2010;Cai et al., 2012).
Therefore, the objective of the research was to know the potentiality of chitosan and yeast elicitor in suppressing seed-borne fungi isolated from bottle gourd, sweet gourd, snake gourd, wax gourd and cucumber.

Materials and Methods
The experiments were conducted in the Laboratory of Biosignaling, Bioactive Compounds and Bio formulation, Plant Disease Diagnostic Clinic (PDDC), Department of Plant Pathology and Seed Pathology Centre (SPC), Bangladesh Agricultural University, Mymensigh-2202 during the period from October, 2015 to November, 2016.
Seeds of bottle gourd, sweet gourd, snake gourd, wax gourd and cucumber were collected from the farmers of Mymensingh districts.These seeds were stored in ziplock bags in refrigerator for further studies.
Blotter method was followed according to ISTA rules for seed health testing (ISTA, 2006) for detection of seed-borne fungi.After 7 days each individual incubated seed was observed under stereo-binocular microscope at 16x and 25x magnifications in order to record the incidence of seed borne fungi.For proper identification of fungi, temporary slides were prepared from the fungal colony and observed under compound microscope and identified with the help of keys suggested by Ellis (1971) and Neergard (1979).
For Chitosan (0.3% solution) preparation, 3g Chitosan was dissolved in concentrated (98%) acetic acid diluted by water to a volume of 1000 mL and 3000 ppm Chitosan stock solution was prepared.From the stock solution 200 ppm, 500 ppm, 1000 ppm & 2000 ppm chitosan solutions were prepared.For Yeast Elicitor (0.3% solution) preparation, Yeast (Saccharomyces cerevisiae) was cultured in YEPDA broth (Yeast extract 1%, Peptone 2% and Dextrose 2%).Erlenmeyer flask (250 mL) having all the ingredients was incubated with shaking on an orbital platform shaker at 30 o C and 140 rpm for 72 hrs for collecting filtrate from the yeast.After 72 hrs the broth was filtrated and filtrate were collected and mixed with ethanol solution following the key of Ari and Cakir (2009).By this procedure 3000 ppm Yeast Elicitor stock solution was prepared.From the stock solution 200 ppm, 500 ppm, 1000 ppm & 2000 ppm Yeast Elicitor solution were prepared.

Seed priming with Chitosan and Yeast Elicitor
Seeds were dipped in respected Chitosan and Yeast Elicitor solution for 2 hrs at room temperature and then seeds were placed in Blotting Paper following ISTA rules for seed testing (ISTA, 2006).As a positive control, seed treatment with Vitavax-200 WP was carried out following the method of Islam et al. (2001).

Statistical analysis
The data were analyzed following completely randomized design (CRD) with three replications by using the M-STAT C statistical software.

Effect of Seed Priming with Chitosan and Yeast
Elicitor on the Association of Seed-borne Fungi Total twelve genera of seed borne fungi were observed associated with tested seeds of cucurbits.Fusarium, Macrophomina, Colletotrichum, Aspergillus, Curvularia, Botrytis, Rhizopus, Phoma, Alternaria, Penicillium, Chaetomium and Cercospora were predominantly associated at various intensity with most of the seed samples (Table 1-5).

Discussion
The present investigation has been carried out by seed priming of vegetable seeds with different elicitors such as Chitosan and Yeast Elicitor.Altogether fourteen fungi, representing twelve genera were recorded in the seeds of bottle gourd, sweet gourd, snake gourd, wax gourd and cucumber collected from seed traders of Mymensingh districts.
In Bottle gourd seeds, eight fungi were detected.Seed priming by Chitosan @ 2000 ppm and Yeast elicitor @ 1000 ppm & 2000 ppm and seed treatment with 0.35% Vitavax-200 WP significantly reduced seed-borne fungal pathogens followed by Chitosan @ 1000 ppm & 500 ppm.In Sweet gourd seeds, ten fungi were detected.Seed priming with Chitosan @ 2000 ppm, Yeast Elicitor @ 2000 ppm and Vitavax-200 WP significantly reduced seed-borne fungal pathogens followed by Yeast Elicitor @ 500 ppm, 1000 ppm & 2000 ppm.The present findings was supported by Begum and Momin (2000); Kamble et al. (1999) where they also reported the association of similar fungi viz.Aspergillus flavus, Penicilium spp., Fusarium spp., Rhizopus spp. with vegetable seeds.In Snake gourd seed, eight fungi were detected.Seed priming by Chitosan @ 2000 ppm significantly reduced seed-borne fungal pathogens followed by Yeast Elicitor Solution @ 200 ppm, 500 ppm, 1000 ppm & 2000 ppm.In Wax gourd seed, ten fungi were detected.Seed priming by Chitosan @ 2000 ppm significantly reduced seed-borne fungal pathogens followed by Chitosan @ 1000 ppm & Yeast Elicitor @ 2000 ppm.The present findings was supported by Begum and Momin (2000) where they also reported the association of similar fungi such as Aspergillus flavus, Penicilium spp., Fusarium spp., Rhizopus spp. with vegetable seeds.In Cucumber, eleven fungi were detected.Seed priming by Yeast Elicitor @ 2000 ppm significantly reduced seed-borne fungal pathogens followed by Chitosan @ 2000 ppm.The present findings was supported by Nasreen and Sultana (2000); Kamble et al. (1999) and Braccini and Dhingara (1996) where they also reported the association of similar fungi viz.

Conclusion
Chitosan and Yeast Elicitor are potential compounds for priming of seed to control seed borne fungi of Cucurbits.
Chitosan and yeast elicitor are bio-polymers and not harmful for ecosystem and completely safe for human health.Therefore, application of chitosan and yeast elicitor can be utilized as seed-treating bio-polymer in replace to chemical pesticides.The effectiveness of these products should also be assessed against seedborne fungi of cucurbits for their commercial application.

Table 2 . Effect of Chitosn and Yeast Elicitor on prevalence of seed-borne fungi of sweet gourd
Values within the same column having a common letter(s) do not differ significantly (P≥0.01)CS indicates Chitosan Solution, YES indicates Yeast Elicitor Solution Figures in the parentheses are arcsine transformed values

Table 5 . Effect of Chitosn and Yeast Elicitor on prevalence of seed-borne fungi of cucumber
Al-Tawaha et al. (2011)totrichum sp., Fusarium spp., Alternaria spp., Macrophomina phaseolina, Aspergillus niger, Penicillium spp.andRhizopus spp.with vegetable seeds.Present investigation indicates that seed priming of cucurbits by elicitor can suppress the growth of seedborne fungi as the elicitor may induce resistance against pathogens.All doses of Chitosan solution shown reduction in seed-borne fungal infection in bottle gourd, sweet gourd, snake gourd, wax gourd and cucumber.Chitosan (2000 ppm) significantly reduced the seedborne fungal pathogens.The findings of the present investigation are in agreement withZheng et al. (2012), who reported that, chitosan coating increased seed germination, plant growth and soybean yield efficiently.Tingda et al. (1994)reported that, 0.1% chitosan help for the growth stimulation of cotton and maize seeds.Alam et al. (2014)also reported that, 1% chiosan solution stimulate the germination percentage of chili seed and control seed-borne fungi associated with chilli seed.Similarly, seed priming with Yeast Elicitor solution showed that all doses of Yeast Elicitor solution reduced seed-borne fungal infection of cucurbits Yeast Elicitor (2000 ppm) significantly reduced the seed-borne fungal pathogens which is statistically similar to the 0.35% Vitavax-200 WP.Al-Tawaha et al. (2011)reported that foliar application of yeast extract hold promises for increasing the seed yield and isoflavone content of soybean seeds.Moreover, due to natural sources both elicitors are environmentally safe.