Strategies for management of fungicide resistance in tomato leaf blight pathogen Alternaria solani in 10 districts of Western Maharashtra in India

Development of fungicide resistance in fungal plant pathogen is of great concern for the management of disease and the losses caused by it. The problem of management of fungicidal resistance is aggravated by the development of cross resistance in plant pathogen against the other fungicides applied in the control of the disease. Tomato leaf blight caused by Alternaria solani is an important disease in 10 districts of western Maharashtra in India. To manage this disease, the tomato growers used at least 8 different fungicides, out of which most of these are proved ineffective in one or other districts due to development of fungicidal resistance in the pathogen and further development of cross resistance to other used fungicides. In this scenario, the present paper discusses the strategies for management of fungicide resistance in tomato leaf blight pathogen Alternaria solani in 10 districts of western Maharashtra in India.


Introduction
Tomato leaf blight disease pathogen Alternaria solani is a most widely distributed pathogen in the cultivation of tomato crop around the world and causes losses up to 78 percent [1]. The disease pathogen in the field persist over season after season and year after year due to the fact that the pathogen in different countries had developed resistant to different fungicides [2][3]. Therefore, for the management of this pathogen certain strategies have to be adopted because it had also developed cross resistant to other fungicides applied in the field for the control of this pathogen [4]. In 10 districts of western Maharashtra, in India, at least 8 different fungicides viz. Dithane M-45, Blitox, Captaf, Bavistin, Kavach, Score, Ridomil and Nativo are applied in the management of this disease, where this pathogen has developed resistant to some fungicides in some districts while to other fungicides in other districts. The knowledge of this fungicide resistance and its cross resistance to other applied fungicides is important to plan the strategies for its management. In this paper we discuss the fungicide resistance and cross resistance developed in Alternaria solani in 10 districts of western Maharashtra and derived the strategies for its managements.

Material and methods
Ten districts of Western Maharashtra viz. Satara, Pune, Nashik, Ahmadnagar, Jalgaon, Sangli, Dhule, Solapur, Nandurbar and Kolhapur were covered for the detection of fungicide resistance development in tomato leaf blight pathogen Alternaria solani against the 8 widely used fungicides viz. Dithane M-45, Blitox, Kavach, Ridomil, Nativo, Bavistin, Captaf and Score. Total one hundred fields (10 fields in each district x 10 districts) having leaf blight infection (Fig 1) and sprayed with either one or more than one of these fungicides were selected to determine the development of fungicide resistance and cross resistance to other used fungicides in Alternaria solani.

Figure 1
Alternaria leaf blight infected tomato Field sprayed with fungicide for its control.

Assessment of fungicide resistance developed in Alternaria solani leaf blight pathogen in the fungicide sprayed tomato crop
The assessment of fungicide resistance in Alternaria solani, the causal agent of leaf blight of tomato, in the fungicide sprayed tomato crop was carried out by isolation of fungicide resistant Alternaria solani isolates from the fungicide sprayed tomato diseased leaves on the fungal growth medium potato dextrose agar (PDA) amended with the corresponding fungicide. To isolate the fungicide resistant Alternaria solani pathogen, the PDA medium was sterilized and amended with the respective corresponding fungicide at the rate of applicable dose in the field (Dithane M-45@0.25%, Blitox@0.25%, Bavistin@0.1%, Captaf@0.1%, Score@0.1%, Kavach@0.25% , Ridomil@0.25%, Nativo@0.25% ). The fungal pathogen was isolated on such medium by employing routine leaf tissue placing method. The isolation plates were incubated in BOD incubator chamber at 28±2 0 C for 7 days. The growth of the pathogen on such fungicide amended media was recorded. The development of Alternaria fungal growth from the infected leaf tissues in the fungicide amended isolation plates indicate the fungicide resistance Alternaria while the absence of fungal growth on such fungicide amended plates indicate the fungicide sensitive Alternaria without development of resistance (Fig 2).
A. fungal growth on FAM B. No fungal growth on FAM The pathogenicity of the fungicide resistant Alternaria isolates were carried out on healthy tomato plant in glass house by spraying the fungal culture on the tomato leaves. The sprayed plants were maintained at controlled temperature of 28 0 C with 97 percent humidity. The symptoms development was observed after 10 days of incubation on the leaves to assess the pathogenicity of the fungicide resistant isolates (Fig 3).
A. B.

Assessment for cross resistance of fungicide resistance Alternaria solani to other fungicides (sprayed in the same tomato fields)
The assessment for cross resistance of fungicide resistant Alternaria solani to other fungicides sprayed in tomato fields were carried out by employing poison food technique method as routinely followed [5]. The sterilized PDA plates amended with individual fungicide were used for this purpose. The growth developed of particular fungicide resistant isolate of A. solani in the form of fungal disc was transferred on PDA plates amended with another fungicide (sprayed in the same tomato field @concentration mentioned above), and the plates were incubated in BOD incubator at 28±2 0 C temperature for 7 days to record the growth. The spread of the fungus from the fungal disc was considered as the presence of cross resistance in the fungicide resistant isolate, whereas no further growth from the fungal disc was considered as fungicidal sensitivity of the isolate and absence of cross resistance to the test fungicide in the isolate ( Fig  4). The non-availability/absence of cross resistance to the test fungicide in the particular fungicide resistant isolate was used in formulating the strategies for the management of fungicide resistant infectious A. solani in the tomato in a particular area/ district of the state.
All the fungicide resistant isolates of Alternaria solani in our studies developed less fungal growth in the fungicidal environment i.e.in the PDA growth medium amended with fungicide than the non -fungicidal environment i.e.in PDA growth medium without fungicide. The decreased growth in the range of 3 to 4 cm was observed in the fungicide amended medium than in the simple PDA growth media. However, there was no variation in other colony characters like coloration of Alternaria colonies.
District-wise pattern for developed fungicide resistant/ availability of fungicidal sensitivity in the pathogen was derived (table 2) for the 8 fungicides used in the management of the disease across the western Maharashtra. In Pune district the A. solani pathogen was observed to develop resistant for all the applied fungicides while in other districts it developed fungicidal resistant to some fungicide and still the fungicidal sensitivity was available for other fungicides. The fungicidal sensitivity present in A. solani in different districts range from 1 to 4 fungicides which appears promising in the management of the pathogen. However, the cross-resistance availability in the pathogen may play an important role in the failure of the disease management. Cross-resistance, is a phenomenon wherein a pathogen carrying resistant to one antimicrobial compound also resists to one or several other antimicrobial compounds, and is one of major threat to human health and sustainable food production. It usually occurs among antimicrobial compounds sharing the same mode of action. Our results on cross resistance studies (   [14] reported triazole fungicides induce cross-resistance to medical triazoles in Aspergillus fumigatus. Heaney et.al [11] reported the strobilurin fungicides and non-strobilurins, famoxadone and fenamidone, form a new cross-resistance group designated as Qol-STAR by FRAC and the resistance had developed rapidly to this group in Erysiphe graminis f.sp.tritici and Sphaerotheca fuliginea. Li-Na Yang et.al [15] stated that pathogen Alternaria alternata could develop cross-resistance to fungicides with different modes of action as indicated by a strong positive correlation between mancozeb and difenoconazole tolerance to A. alternata. They also found a positive association between mancozeb tolerance and aggressiveness of A. alternata, suggesting no fitness penalty of developing mancozeb resistance in the pathogen and hypothesize that mechanism such as antimicrobial compound efflux and detoxification that limit intercellular accumulation of natural/synthetic chemicals in pathogen might account for the cross-resistance.

Conclusion
Tomato leaf blight caused by fungal pathogen Alternaria solani is an important disease in 10 districts of western Maharashtra in India. To manage this disease, the tomato growers used at least 8 different fungicides, out of which most of these are proved ineffective in one or other districts due to development of fungicidal resistance in the pathogen and further development of cross resistance to other used fungicides. The fungicide resistance developed in the pathogen for Dithane M-45, Blitox, Kavach and Ridomil can be managed by application of Nativo and Score fungicide while the fungicide resistant developed for Nativo, Captaf and Score fungicide in the pathogen can be managed by Dithane M-45 fungicide. The Bavistin resistant isolate had developed cross-resistant to all the fungicide used and therefore Bavistin should not be used in the management of this disease pathogen. These results will be helpful to the farmers in planning the management strategies for the control of this most important disease of tomato and for the research workers to carry out basic studies in the fungicide resistance and cross resistance.

Compliance with ethical standards
No experimental animals are used in these studies.