Introduction

The grape (Vitis vinifera) is one of the most economically important fruit crops in the world¹. Anthracnose is one of the most damaging diseases of grape and is caused by Gloeosporium ampelophagum (Pass.) Sacc. and responsible for yield losses in commercial grape production. In wet humid regions the disease incidence and severity on various cultivars of grape can be very serious². Infection may occur on all succulent plant material but is most common on fruit and shoots. Lesions on berries are initially small, circular and reddish in color. Acervuli are also produced in these lesions. Leaf spots are often numerous and resemble those on fruit. The center often drops out leaving a shot hole appearance. Young leaves are more susceptible than older leaves and are malformed when veins become infected. Fungicides have been extensively used to control anthracnose of grape, but cause environmental pollution and leave residues in the agricultural soil and on products. Chemical usage has been effective, although resistance to these fungicides is developing. The development of carbendazim resistance against G. ampelophagum in Maharashtra³ and other States of India was studied by many workers^{4 -10}.

Materials and Methods

Sensitivity of pathogen

The infected samples of grapes were collected from different districts known as ‘grape belt’ of Maharashtra like Ahmednagar, Nashik, Pune and Solapur during 2009 & 2010 crop seasons. Isolation of pathogen was done by inoculating the samples on Czapek-Dox agar medium. The cultures were further purified and maintained on same medium at 27± 1° C. A total of 37 isolates were purified and tested against carbendazim fungicide by ‘Poisoned Food Technique’¹¹ to check their sensitivity. Czapek-Dox agar medium (2X) was prepared and it was then sterilized and 10 ml of this was properly mixed with 10 ml of fungicide (2X a.i. concentrations) selected for study in sterile Perti plates. A series of concentrations was prepared; the fungicide was thoroughly mixed with medium and allowed to solidify. A 4 mm disc of the fresh grown G. ampelophagum isolates was transferred aseptically at the centre of Petri plate. On the basis of minimum inhibitory concentration (MIC) the resistant and highly resistant population was calculated by multiplying four times to the sensitive baseline dose as per guidelines given by fungicide resistance action committee (FRAC). The data of radial growth was analyzed for MIC and essential dose (ED ₅₀) by using following equation¹².

(Where, Y = radial growth as percentage of control, H= upper limit of curve, Exp= logarithmic exponent, a= regression constant, b= regression coefficient and x= measured points).

Study of Passage

In order to study the effect of passage in vitro wild sensitive isolate GA-9 in each passage was cultured on agar plates containing sub-lethal dose of carbendazim (0.3 μg/ml). The plates without fungicide served as control. A 4 mm diameter disc of freshly grown culture taken from the culture of previous passage of the same isolate was placed at the centre of each plate. In each passage linear growth was measured after eight days. Percentage increase of growth of the isolate from passage to passage was considered as increase in carbendazim resistance or vice-versa. The development of resistance thus was studied up to 5^th passage. Alternate passage carbendazim with triadimefon, metalaxyl, difenoconazole, myclobutanil, propiconazole and mixed passage with the same fungicide were also carried out.

Passage studies were also carried out on the grape berries (Vitis vinifera L. var. Thomson seedless). The grape berried were inoculated with spore suspension taken from the culture of previous passage of the same isolate was inoculated on grape berries. The concentration of carbendazim and wild sensitive isolate GA-9 was kept same as used in vitro studies. At each passage percent disease index (PDI) was calculated. PDI increased from passage to passage considered as increase in the carbendazim resistance. The development of resistance was studied up to 5^th passage both in alternate and in mixture of different fungicides.

Results and Discussion

Results in Table 1 indicate that in vitro individually culturing of the pathogen in carbendazim increased growth significantly up to 5^th passage. However, alternate culturing of pathogen on difenoconazole and myclobutanil reduced growth significantly. This reduction was more prominent with myclobutanil than other fungicides. Interestingly there was also significant reduction in the growth of pathogen when cultured on the carbendazim in combination with difenoconazole and myclobutanil on grape berries (Table 2). In vivo results are given in Table 3 and 4. It was seen that again treatment of carbendazim to grape berries for five successive passages increased PDI on grapes. However, treatment of carbendazim alternately with triadimefon, metalaxyl, difenoconazole, myclobutanil and propiconazole reduced PDI significantly. Use of carbendazim with difenoconazole, myclobutanil and propiconazole were most useful in controlling grape anthracnose. There are some evidences for increase of resistance due to continuous exposure of pathogen to the fungicides^{12, 13}. Alternate or mix application of fungicide must have different mode of action¹⁴ and in the present investigation there might have less chances to mutate or to adopt resistance in G. ampelophagum due to use of other group of fungicides. These results also agree with the earlier work in case of Septoria nodorum against carbendazim¹². Similar results were obtained by author in Penicillium digitatum against thiophanate-methyl and in Alternaria alternata against aureofungin^15,16 causing fruit rot of grapes.

Table 1: Effect of continuous exposure to carbendazim and to carbendazim alternately with other different fungicide on growth of Gloeosporium ampelophagum on agar medium during five successive passages.

Table 2: Effect of continuous exposure to carbendazim and to carbendazim alternately with other fungicides on growth of Gloeosporium ampelophagum on grape berries during five successive passages.

Table 3: Effect of continuous exposure to carbendazim and to carbendazim mixed with other different fungicide on growth of Gloeosporium ampelophagum on agar medium during five successive passages.

Table 4: Effect of continuous exposure to carbendazim and to carbendazim mixed with other different fungicide on growth of Gloeosporium ampelophagum on grape berries during five successive passages.

Acknowledgement

Authors are grateful to the University Grants Commission, New Delhi for providing financial support in the form of major research project to carry out these investigations. Thanks are also due to the Principal, Vasantrao Naik College for providing the facilities to complete this work.

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