Recent Advances on the Use of Natural and Safe Alternative Method to Control Grey Mould of Grapes

Grey mould of grapes ( Vitis vinifera L var. red globe ) caused by Botrytis cinerea Pers. ex. Fr. is recorded as an economically serious pathogen found in stored and imported grapes in India and which may cause heavy loss in all grape producing countries. Therefore the management of grey mould is equally important. In conventional agriculture the application of fungicides to grapes after harvest to reduce decay has been increasingly, curtailed by the development of pathogen resistance, the lack of knowledge of fungicide, negative public approach regarding the safety of pesticides and consequent restrictions on fungicide use. The study of alternative methods to control of grey mould decay has developed over several decades. Twenty five isolates of B.cinerea were isolated from infected samples of grape collected from various localities of Maharashtra. Out of which isolate Bc -19 was highly sensitive (290.8  g/ml) while isolate Bc- 11 was resistant (1095.1  g/ml). Sensitivity of B. cinerea was tested using thiophanate methyl by food poisoning methods. Experiments were performed in Research Laboratory, KVP to find out the natural and safe alternative method to control of resistant Bc -11. Six fungicides viz. dithane M-45, thiram, zineb, benomyl, carbendazim and captafol were used individually and in mixture with thiophanate methyl. Benomyl gave a significant PCE individual at 50 µg/ml (70.01) and at 100 µg/ml PCE value (83.12) when compared with thiophanate methyl at 100 µg/ml (58.12), Captafal 50 µg/ml and 100 µg/ml (64.18), zineb 50 µg/ml (62.13) and 100 µg/ml (75.15) PCE values, thiram at 50 µg/ml (54.12) and 100 µg/ml (PCE- 65.10) and dithane M-45 at 50 µg/ml PCE (38.02) and 100 µg/ml (48.15) PCE values. Dithane and thiram was less effective. Trichoderma viride was found to be the most effective in reducing the colony diameter of B. cinerea (69.89 - 68.12). Thus T. viride proves better and safe for the management of B. cinerea of grape as a


Introduction
The most postharvest decay of table grapes (Vitis vinifera L var. red globe) caused by Botrytis cinerea Pers ex.Fr. is recorded as an economically serious pathogen found in stored and imported grapes in India and which may cause heavy loss in all grape producing countries. It is an apportunistic pathogen on a wide variety of crops, causing a disease known as grey mould through infections via wound [11]. The world wide post harvest loss is great and immense due to B. cinerea ranged between 10 to 50%.The use of chemical becomes essential when use of resistant varieties, fungal cultural practices or alternation of the environment is inadiequate to manage this pathogen. Nowdays, chemicals play role in crop protection [9,10]. In cold stored grapes (-1º C), the main decay pathogens are B.cinerea Pers., Cladosporium herbarum Link., Alternaria alternata Keissler, Rhizopus stolonifer Ehrenb, Aspergillus niger Bainer and Penicillium expansum Link. Further losses occur during grading, packing, transport and finally marketing as fresh produce. Among these pathogens B.cinerea Pers. is most trouble because of its vigrous growth rate and ability to spread among grapes [7,8]. However, the use of systemic fungicides are not allowed on grapes and there are incresing regulatory restrictions on the use of chemical fugicides.
The use of alternative method to control postharvest decay of table grape extensively reviewed. Therefore minimize the economic losses and improve the quality of grapes. To develop management strategies to reduce fungicide used and adopt natural and safe alternative method to control the grey mould of grape. Wherein biological control would be significant [3]. Trichoderma is known to play an important role in natural and safe alternative because of it is easy to isolate and culture, grow rapidly on many medium and affect of a wide range of plant pathogen [1,2,4].
Present investigation was carried out to evaluate a number of Trichoderma species and isolate, alone and in combination with different fungicides viz. benomyl, dithane, zeneb, thiram and captafol for the controlling B.cinerea. Infected samples were collected from different localities of Maharashtra in 2008 to 2011. The infected samples were surface sterilized with 0.1% HgCl 2 solutions for one minutes with gentle agitation. Samples were rinsed with sterile distilled water (SDW) up to 5 to 6 times then cut into smaller pieces and place aseptically at equidistant onto sterile potato dextrose agar (PDA) augmented with streptomycin (20 µg/ml) in petriplates. Plates were incubated at 22 ±2º C in dark for seven days. The pure culture of the pathogen was obtained. The pathogen was purified using PDA medium. Seven days old culture of B. cinerea were used for the experiement. Sensitivity of B. cinerea was tested using thiophanate methyl by food poisoning methods [6]. Isolate Bc-11 was resistant while isolate Bc-19 are sensitive were obtained. Resistant Bc-11 was used for the experiement ( Table 2).

Isolation of Trichoderma spp.
Three isolates of Trichoderma were isolated from mangrove soil of Thane creek. Approximately 20 gms of soil were added to 100 ml of SDW and shaking well for 15 min. The resultant suspension used to inoculate in PDA. All plates were incubated at room temperature 22±2º C. The fungal colonies appearing after 7 days were picked and restreaked until pure culture were obtained. The fungal colonies were identified and used against B.cinerea. PDA broth pour in 250 ml conical flask and inoculate the T.viride. and T.harzianum and inoculate at 22±2º C. After seven days T.viride. and T.harzianum were separated from the broth mycelial mats were used. Dried mycelial mat were grinded in powder form. Powder of Trichoderma and fungicides were used against B.cinerea in vitro.  (Fig 3). Six fungicides viz. dithane M-45, thiram, zineb, benomyl, carbendazim and captafol were used individually and in mixture with thiophanate methyl. The concentration was used at 50 µg/ml and 100 µg/ml in potato dextrose agar medium (PDA). After 6 days growth of B.cinerea was observed and percentage control efficacy (PCE) was measured (  (Table 3).

Results and Discussions
Fungicides were mix with thiophanate methyl in PDA. Test fungus inoculated at the centre of petriplate, after 6-7 days colony diameter was measured and PCE value determined. PCE was highly increased with benomyl, carbendazim, captafol, zineb, thiram and dithane M-45. It was going upto 42.08 -75.88 at 50 µg/ml concentration while PCE values ranged from 60.10 -86.12 at 100 µg/ml concentration. Benomyl use as systemic fungicide PCE 75.88 as 50 µg/ml and 86.12 at 100 µg/ml as compared with individually and in mixture with thiophanate methyl PCE value appeared more fruitful and fallowed by other fungicides.  Values are the mean of three replicates. Table 4 revealed that the effects of the three species of Trichoderma that had the greatest potential of antifungal activity. T. viride and T. harzianum was found to be the most effective in reducing the colony diameter of B. cinerea (69.89 -68.12). It also reported that T. viride and T. harzianum significantly suppressed Fusarium udam is soil and roots of pigeon pea [9]. Reference [8] observed that the isolate of T. virens and T. viride were the most effective in which maximum growth inhibition of mycelium of Fusarium oxysporium was recorded i.e., 77.7% and 77.55%. Reference [5] reveals that Trichoderma emerged as most potential bioprotectants for management of wide variety of plant diseases. Table 5 revealed that all treatment significantly reduced the inhibition zone of B.cinerea. The treatment with powder of Trichoderma and fungicides (dithane M-45, carbendazim, thiophanate methyl, antracol and benomyl) gave overall steady results.  viride, T. harzianum with thiophanate methyl was beneficial in controlling of this disease but not more effective.
Research during the previous five decades indicates another potential option for fruit disease management. That is the biological control of fruit diseases. Bio-control assumes special significance in being an eco-friendly and cost-effective strategy that should be used in integration with other strategies for a greater level of protection with sustained fruit production.