Carbendazim: a Potent Fungicide with Anticancer Therapeutic Properties

Article i

Review Anticancer Agents Med Chem

. 2018;18(1):38-45. doi: 10.2174/1871520616666161221113623.

Double Edge Sword Behavior of Carbendazim: A Potent Fungicide With Anticancer Therapeutic Properties

Karan Goyal 1, Ajay Sharma 2, Ridhima Arya 1, Rohit Sharma 1, Girish K Gupta 3, Anil K Sharma 1

Affiliations expand

PMID: 28003000 DOI: 10.2174/1871520616666161221113623

Abstract

Background: A number of benzimidazole derivatives such as benomyl and carbendazim have been known for their potential role as agricultural fungicides. Simultaneously carbendazim has also been found to inhibit proliferation of mammalian tumor cells specifically drug and multidrug resistant cell lines.

Objective: To understand the dual role of Carbendazim as a fungicide and an anticancer agent, the study has been planned referring to the earlier studies in literature.

Results: Studies carried out with fungal and mammalian cells have highlighted the potential role of carbendazim in inhibiting proliferation of cells, thereby exhibiting therapeutic implications against cancer. Because of its promising preclinical antitumor activity, Carbendazim had undergone phase I clinical trials and is under further clinical investigations for the treatment of cancer. A number of theoretical interactions have been pinpointed. There are many anticancer drugs in the market, but their usefulness is limited because of drug resistance in a significant proportion of patients. The hunger for newer drugs drives anticancer drug discovery research on a global platform and requires innovations to ensure a sustainable pipeline of lead compounds.

Conclusion: Current review highlights the dual role of carbendazim as a fungicide and an anticancer agent. Further, the harmful effects of carbendazim and emphasis upon the need for more pharmacokinetic studies and pharmacovigilance data to ascertain its clinical significance, have also been discussed.

Keywords: Anticancer; benzimidazole; carbendazim; fungicide; sword.; therapeutic.

Article Ii

Toxicity, monitoring and biodegradation of the fungicide carbendazim

Article (PDF Available) in Environmental Chemistry Letters 14(3) · June 2016 with 4,340 Reads

DOI: 10.1007/s10311-016-0566-2

Cite this publication

Simranjeet Singh

26.04Lovely Professional University

Nasib Singh

+ 4

Vijay Kumar

25.48Regional Ayurveda Research Institute for Drug Development, Ministry of AYUSH, Govt of India

ShivikaDatta

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Abstract

The increasing use of toxic pesticides is a major environmental concern. Carbendazim is a systemic fungicide having wide applications for controlling fungal diseases in agriculture, forestry and veterinary medicines. Carbendazim is a major pollutant detectable in food, soil and water. Carbendazim extensive and repeated use induces acute and delayed toxic effects on humans, invertebrates, aquatic life forms and soil microorganisms. Here, we review the pollution, non-target toxicity and microbial degradation of carbendazim for crop and veterinary purposes. We found that carbendazim causes embryotoxicity, apoptosis, teratogenicity, infertility, hepatocellular dysfunction, endocrine-disrupting effects, disruption of haematological functions, mitotic spindle abnormalities, mutagenic and aneugenic effect. We also found that carbendazim disrupted the microbial community structure in various ecosystems. The detection of carbendazim in soil and reservoir sites is performed by spectroscopic, chromatographic, voltammetric, nanoparticles, carbon electrodes and mass spectrometry. A review of the degradation of carbendazim shows that carbendazim undergoes partial to complete biodegradation in the soil and water by Azospirillum, Aeromonas, Alternaria, Bacillus, Brevibacillus, Nocardioides, Pseudomonas, Ralstonia, Rhodococcus, Sphingomonas, Streptomyces and Trichoderma.

Article I

Toxicity, monitoring and biodegradation of the fungicide carbendazim

Article (PDF Available) in Environmental Chemistry Letters 14(3) · June 2016 with 4,317 Reads

DOI: 10.1007/s10311-016-0566-2

Cite this publication

Simranjeet Singh

26.04Lovely Professional University

Nasib Singh

+ 4

Vijay Kumar

25.48Regional Ayurveda Research Institute for Drug Development, Ministry of AYUSH, Govt of India

ShivikaDatta

Show more authors

Abstract

Article Ii

Review

Published: 01 June 2016

Toxicity, monitoring and biodegradation of the fungicide carbendazim

Simranjeet Singh, Nasib Singh, Vijay Kumar, ShivikaDatta, Abdul BasitWani, Damnita Singh, Karan Singh &Joginder Singh

Environmental Chemistry Letters volume 14, pages317–329(2016)Cite this article

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Abstract

J Toxicol Environ Health A    . 2004 Oct 8;67(19):1501-15. doi: 10.1080/15287390490486833. Endocrine-disrupting activity in carbendazim-induced reproductive and developmental toxicity in rats

Shui-Yuan Lu 1, Jiunn-Wang Liao, Min-Liang Kuo, Shun-Cheng Wang, Jenn-Sheng Hwang, Tzuu-Huei Ueng Affiliations expand

 PMID: 15371226

 DOI: 10.1080/15287390490486833 Abstract

This study was designed to investigate the endocrine-disrupting activity of carbendazim- induced reproductive and developmental toxicity in Sprague-Dawley rats treated orally with the fungicide. Cotreatment of male rats with 675 mg/kg carbendazim and 50 or 100 mg/kg flutamide, an androgen receptor antagonist, once daily for 28 d blocked decrease of testis weight induced by treatment with carbendazim alone. The cotreatment prevented losses of spermatozoa and cell morphology and decrease of sperm concentration induced by carbendazim. Premating treatment of male and female rats with 200 mg/kg carbendazim for 28 d produced androgenic effects including incomplete development of uterine horn, enlargement of uretha, absence of vagina, and induction of seminal vesicles in female offspring, without marked effects in male offspring. Premating treatment with 100mg/kg benomyl, the parent compound of carbendazim, resulted in incomplete development of uterine horn and absence of vagina in female offspring and produced testis and epidydimis atropy in male offspring. Treatment of male rats with 25, 50, 100, 200, 400, and 800 mg/kg carbendazim for 56 d produced dose-dependent increases of androgen receptor concentrations in testis and epididymis. Additions of 5, 50, and 500 microM carbendazim to testis extract from untreated rats replaced binding of [3H]-5 alpha-dihydrotestosterone to androgen receptor in a concentration-dependent manner. The present study demonstrates that reproductive toxicity induced by carbendazim is blocked by an androgen receptor antagonist in male rats and developmental toxicity of the fungicide shows androgenic properties in female offspring. These results suggest that androgen- and androgen receptor-dependent mechanisms are possibly involved in carbendazim-induced toxicity.

Toxicology. 1989 Jul 17;57(2):173-82.

doi: 10.1016/0300-483x(89)90163-7. Effects of the benomyl metabolite, carbendazim, on the hypothalamic- pituitary reproductive axis in the male rat

J M Goldman 1, G L Rehnberg, R L Cooper, L E Gray Jr, J F Hein, W K McElroy

Affiliations expand

 PMID: 2501910

 DOI: 10.1016/0300-483x(89)90163-7 Abstract

Carbendazim (MBC), the bioactive metabolite of the fungicide benomyl, has been reported to induce a number of testicular alterations in male rats. Since it is possible that extragonadal changes contribute to the appearance of such effects, the present study focused on the presence of concurrent endocrine changes in the hypothalamic and pituitary components of the brain-pituitary-testicular axis. Subchronic administration of MBC (50, 100, 200 or 400 mg/kg) was found to cause a dose-related elevation in serum follicle stimulating hormone (FSH) and pituitary luteinizing hormone (LH). Values for prolactin and thyroid-stimulating hormone remained unchanged. No statistical differences in gonadotropin-releasing hormone concentrations were present in mediobasal hypothalamus, although an elevation in anterior hypothalamic values was found at the low dose, followed by a dose-related decline. These findings demonstrate that previously reported gonadal differences following subchronic exposure to carbendazim are accompanied by alterations elsewhere in the reproductive system which appear to involve both changes in Sertoli cell-pituitary feedback signals and direct effects of the compound on the central nervous system. 1) Carbendazim

6-Gingerol-rich fraction from Zingiber officinale ameliorates carbendazim induced endocrine disruption and toxicity in testes and epididymis of rats. Salihu M1, Ajayi BO1, Adedara IA1, de Souza D2, Rocha JBT2, Farombi EO1.

This study evaluated the protective effects of 6-gingerol-rich fraction (6-GRF) from Zingiber officinale on carbendazim (CBZ)-induced reproductive toxicity in rats. Adult male rats were treated with either CBZ (50 mg/kg) alone or in combination with 6-GRF (50, 100 and 200 mg/kg) for 14 consecutive days. Gas chromatography-mass spectrometry (GCMS) analysis revealed that 6-GRF consists of ten bioactive chemical components with 6-gingerol being the most abundant (30.76%). Administration of 6-GRF significantly (p < .05) prevented CBZ- mediated increase in absolute and relative testes weights as well as restored the sperm quantity and quality in the treated rats to near control. In testes and epididymis, 6-GRF significantly abolished CBZ-mediated increase in oxidative damage as well as augmented antioxidant enzymes activities and glutathione level in the treated rats. Moreover, CBZ administration alone significantly decreased plasma levels of testosterone, thyrotropin, triiodothyronine and tetraiodothyronine, whereas follicle-stimulating hormone was significantly elevated without affecting luteinising hormone and prolactin levels when compared with the control. Conversely, 6-GRF ameliorated the disruption in the hormonal levels and restored their levels to near normalcy in CBZ-treated rats. Collectively, 6-GRF inhibited the adverse effects of CBZ on the antioxidant defence systems, hormonal balance and histology of the testes and epididymis in rats.

Insights into a Possible Mechanism Underlying the Connection of Carbendazim-Induced Lipid Metabolism Disorder and Gut Microbiota Dysbiosis in Mice (2018) Jin C1, Zeng Z1, Wang C1, Luo T1, Wang S1, Zhou J1, Ni Y1, Fu Z1, Jin Y

Carbendazim (CBZ), a systemic, broad-spectrum benzimidazole fungicide, is widely used to control fungal diseases and has been regarded as an endocrine disruptor that causes mammalian toxicity in different target organs. Here, we discovered that chronic administrations of CBZ at 0.2, 1, and 5 mg/kg body weight for 14 weeks not only changed the composition of gut microbiota but also induced significant increases in body, liver, and epididymal fat weight in mice. At the biochemical level, the serum triglyceride (TG) and glucose levels also increased after CBZ exposure. Moreover, the level of serum lipoprotein lipase (LPL), which plays an important role in fatty acid release from TG, was decreased significantly. For gut microbiota, 16S rRNA gene sequencing and real-time qPCR revealed that CBZ exposure significantly perturbed the mice gut microbiome, and gas chromatography found that the production of short- chain fatty acids were altered. Moreover, CBZ exposure increased the absorption of exogenous TG in the mice intestine and inhibited the TG consumption, eventually leading the serum triglyceride to maintain higher levels. The increase of lipid absorption in the intestine direct caused hyperlipidemia and the multi-tissue inflammatory response. In response to the rise of lipid in blood, the body maintains the balance of lipid metabolism in mice by reducing lipid synthesis in the liver and increasing lipid storage in the fat. Chronic CBZ exposure induced the gut microbiota dysbiosis and disturbed lipid metabolism, which promoted the intestinal absorption of excess triglyceride and caused multiple tissue inflammatory responses in mice.

11.6.2011 EN Official Journal of the European Union L 152/1

II (Non-legislative acts)

REGULATIONS

COMMISSION REGULATION (EU) No 559/2011 of 7 June 2011 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for captan, carbendazim, cyromazine, ethephon, fenamiphos, thiophanate-methyl, triasulfuron and triticonazole in or on certain products (Text with EEA relevance)

THE EUROPEAN COMMISSION, (4) On the basis of additional data submitted by South Africa and Germany, the Authority further refined its earlier evaluation of the consumer exposure for carbendazim ( 3) and thiophanate-methyl ( 4 ). It concluded Having regard to the Treaty on the Functioning of the European that it is necessary to lower the MRLs as regards Union, carbendazim for grapefruits, oranges, and tomatoes and as regards thiophanate-methyl for tomatoes.

Having regard to Regulation (EC) No 396/2005 ( 1) of the European Parliament and of the Council of 23 February 2005 (5) For ethephon ( 5), fenamiphos ( 6 ), triasulfuron ( 7) and triti on maximum residue levels of pesticides in or on food and feed conazole ( 8), the Authority submitted reasoned opinions of plant and animal origin and amending Council Directive on the existing MRLs in accordance with Article 12(2) of 91/414/EEC, and in particular Article 14(1)(a) and Regulation (EC) No 396/2005. The Authority concluded Article 49(2) thereof, that it is necessary to lower the MRLs as regards tria sulfuron for barley, oats, rye, and wheat and as regards fenamiphos for tomatoes, aubergines, peppers, water Whereas: melons, courgettes, Brussels sprouts, bananas, peanuts and oilseeds and to raise the MRL for grapes. As regards triticonazole, the Authority concluded that no MRL needs to be modified. It is appropriate to move (1) For captan, carbendazim, cyromazine, ethephon, fena the MRLs on new commodities for these four substances, miphos, thiophanate-methyl, triasulfuron and triti temporarily set in Part B of Annex III to Regulation (EC) conazole maximum residue levels (MRLs) are set in No 396/2005, to Annex II to that Regulation. Annex II and Part B of Annex III to Regulation (EC) No 396/2005.

(6) Based on the reasoned opinions of the Authority and taking into account the factors relevant to the matter (2) For captan the Commission was informed that uses on under consideration, the appropriate modifications to celery, spinach, and parsley were revoked and thus the the MRLs fulfil the requirements of Article 14(2) of Regu corresponding MRLs could be reduced without requiring lation (EC) No 396/2005. the opinion of the European Food Safety Authority, here inafter ‘the Authority’, in accordance with Article 17 of Regulation (EC) No 396/2005. (7) Through the World Trade Organisation, the trading partners of the Union were consulted on the new MRLs and their comments have been taken into account. (3) For cyromazine an evaluation by the Authority ( 2 ) indicated that the MRL for lettuce may raise concerns 3 of consumer protection. The Authority recommended ( ) EFSA Scientific Report (2009) 289. 4 lowering that MRL. These concerns also apply to scarole. ( ) See footnote 3. ( 5 ) EFSA Journal (2009) 7(10): 1347. ( 6 ) EFSA scientific report (2009) 331. ( 1 ) OJ L 70, 16.3.2005, p. 1. ( 7 ) EFSA scientific report (2009) 278. ( 2 ) EFSA scientific report (2008) 168. ( 8 ) EFSA scientific report (2009) 277. L 152/2 EN Official Journal of the European Union 11.6.2011

(8) A reasonable period should be allowed to elapse before (1) Annex II is amended in accordance with the Annex to this the modified MRLs become applicable in order to permit Regulation. Member States and interested parties to prepare them selves to meet the new requirements which will result from the modification of the MRLs. (2) In Part B of Annex III, the columns for ethephon, fena miphos, triasulfuron and triticonazole are deleted.

(9) Annex II to Regulation (EC) No 396/2005 and Part B of Annex III to that Regulation should therefore be Article 2 amended accordingly. As regards the active substances and the products set out in the following list, Regulation (EC) No 396/2005 as it stood before being amended by this Regulation shall continue to apply to (10) In order to allow for the normal marketing, processing products which were lawfully produced before 1 January 2012: and consumption of products, this Regulation should provide for a transitional arrangement for products which have been lawfully produced before the modifi (a) captan: celery, spinach and parsley; cation of the MRLs and for which information shows that a high level of consumer protection is maintained. (b) carbendazim and thiophanate-methyl: frozen, canned, preserved and processed products of grapefruits, oranges (11) The measures provided for in this Regulation are in and tomatoes; accordance with the opinion of the Standing Committee on the Food Chain and Animal Health and (c) fenamiphos: fruiting vegetables, bananas, oilseeds and neither the European Parliament nor the Council has Brussels sprouts. opposed them,

Article 3 HAS ADOPTED THIS REGULATION: This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the Article 1 European Union. Annexes II and III to Regulation (EC) No 396/2005 are amended as follows: It shall apply from 1 January 2012.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

Done at Brussels, 7 June 2011.

For the Commission The President José Manuel BARROSO 11.6.2011 EN Official Journal of the European Union L 152/3

ANNEX

Annex II to Regulation (EC) No 396/2005 is amended as follows: The columns for captan, carbendazim, cyromazine, ethephon, fenamiphos, thiophanate-methyl, triasulfuron and triti conazole are replaced by the following: L 152/4 EN Official Journal of the European Union 11.6.2011

Triticonazole

0,01 (*) 0,01

Triasulfuron (10)

hohnt-ehl(R) Thiophanate-methyl (9)

fenamiphos) (*) 0,2

sulphoxide and sulphone expressed as as expressed sulphone and sulphoxide (8) Fenamiphos (sum of fenamiphos and its its and fenamiphos of (sum Fenamiphos 0,02 (*) 0,02

Ethephon (7)

0,05 (*) 0,05

Cyromazine (6)

abnai)(R) carbendazim) (*) (*) 0,1 0,02

6 6 6 6 6

benomyl and carbendazim expressed as as expressed carbendazim and benomyl (5)

(*) Carbendazim and benomyl (sum of of (sum benomyl and Carbendazim 0,7 0,2 0,1 0,05 (*) 0,05 0,1 0,1 0,1 0,1 0,2

0,1

Captan (4) 0,2 0,7 ) a Pesticide residues and maximum residue levels (mg/kg) Pesticide residues and maximum residue ‘ 0,1 (*) 0,1 0,05 (*) 0,05 (3) 0,7

0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 0,3

ugli and other hybrids) ugli and Tree nuts (shelled or unshelled) or (shelled nuts Tree Citrus fruit Citrus FRUIT FRESH OR FROZEN; NUTS FROZEN; OR FRESH FRUIT Groups and examples of individual products to which the MRLs apply ( apply MRLs the which to products individual of examples and Groups Oranges (Bergamot, bitter orange, chinotto and other hybrids) mineola), other (except and hybrids) tangelo chinotto other sweeties, orange, and pomelos, bitter mineola lemon) (Shaddocks, 1. (i) (Bergamot, tangerine, (Citron, Grapefruit Oranges (Clementine, Lemons Limes Mandarins Others nuts (ii) nuts Almonds Brazil Cashew (Filbert) Chestnuts Coconuts Hazelnuts (2) (1) Code number 0110010 0110000 0100000 0120020 0120030 0120040 0120050 0120060 0120000 0120010 0110020 0110030 0110040 0110050 0110990 11.6.2011 EN Official Journal of the European Union L 152/5

(10)

(9)

(*) 0,1

(8) 0,02 (*) 0,5 0,5 (**) (**) 0,5 2 (*) 2 0,3 0,1 (*) 0,02 0,5 (*) 0,1 (7) 0,3 3 0,05 (*) (*) 0,05 0,05 (6) 0,05 (*) 0,05 (*) 0,05 (**) (**) 0,05 (*) (*) 0,6 0,05 (*) 0,05 (*) 0,05 (*) (*) 0,05 3 0,05 0,05 0,7 2 (5) (**) (**) 0,2 0,2 0,1 0,1 0,1 0,1 0,5 0,1 0,2 (*) 0,1 0,03 0,02 (*) 0,02 (*) 0,1 (4) 0,2 0,5 0,5 1 0,02 (*) 0,02 5 (3) 0,02 (*) 0,02

0,2 0,2 0,3 0,5 3 (+) 3

3 (+) 3 0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 3 0,02 (*) 0,02

0,02 (*) 0,02 (*) 0,02 0,02 (*) 0,02 Strawberries Table and wine grapes wine and Table Pome fruit Pome Stone fruit Stone Berries & small fruit small & Berries Pine nuts Macadamia Pecans apple) Pine pear) Pistachios Walnuts (Crab cherries) Others (Oriental hybrids) (iii) sloe) Apples sour Pears similar Quinces mirabelle, cherries, Medlar and Loquat greengage, Others (sweet (iv) (Nectarines Apricots (Damson, Cherries Peaches Plums grapes Others grapes (v) (a) Table Wine (b) (2) (1) 0130050 0130990 0151010 0130010 0140010 0130020 0130030 0130040 0140000 0140030 0140990 0120070 0151000 0120080 0120090 0120100 0120110 0120990 0130000 0150000 0151020 0140020 0140040 0152000 L 152/6 EN Official Journal of the European Union 11.6.2011

(10)

(9) 0,1 (*) 0,1 (*) 0,1 (8) (*) 0,02 0,02 (*) 0,02 (**) (**) (**) (**)

(7) 5 (+) 0,05 (*) 0,05 (*) 0,05 (*) 0,05 (*) (*) 0,05 (*) 0,05 (*) 0,05 0,05 (6) 0,05 (*) (*) 0,05 0,02 (*) 0,1 (**) (**) (**) (**) (5) (*) 20 0,05 (*) (**) (**) (**) (**) 0,05 (*) 0,1 (*) (*) 0,05 0,05 (4) 3 (+) 0,02 (*) 0,02 (*) 0,02 0,02 (*) 0,02 3 (+) 3

0,02 (*) 0,02

3 (+) 3 (3) 0,02 (*) 0,02 0,1 (*) 0,1 0,02 (*) 0,02

3 (+) 3 0,02 (*) 0,02 0,02 (*) 0,02 0,1 (*) 0,1 0,02 (*) 0,02 (*) 0,02

(Citrus aurantifolia x Fortunella spp.)) (Citrus aurantifolia arcticus), nectar raspberries (Rubus arcticus x idaeus)) and berries, service hawthorn, buckthorn (sea sallowthorn), other treeberries) Other small fruit & berries & fruit small Other Edible peel Edible Cane fruit Cane Miscellaneous fruit Miscellaneous Dewberries (Loganberries, boysenberries, and cloudberries) species) (Rubus and ribes bramble/raspberry, boysenberries, white) arguta)) other arctic bilberries)) (Loganberries, ash, and (c) (Wineberries, (Actinidia with Blackberries (red Dewberries mountain black Raspberries (Kiwiberry hybrids (Bilberries) Others (Cowberries (appleberry), berry) (red, (d) medlar) (Including Blueberries limequats hips Cranberries chokeberry (arbutus Currants (mediteranean Gooseberries kumquats, Rose (Black Mulberries Azarole nagami Elderberries Others kumquats, (vi) olives (a) Dates (Marumi Figs Table Kumquats (2) (1) 0154060 0154070 0154080 0154990 0161000 0153020 0153990 0154020 0153000 0161040 0153010 0154010 0161010 0153030 0154000 0154030 0154040 0154050 0160000 0161020 0161030 11.6.2011 EN Official Journal of the European Union L 152/7

(10)

(9)

(8) (**) (**) (*) (**) 0,1 (*) (*) 0,1 (*) 0,1 1 1 0,1 (**) (**) (7) 0,1 (*) 0,1 0,05 (*) 0,05 (*) (*) 0,05 (*) 0,05 0,05 (**) (**) (**) (*) 0,05 (*) 0,05 (6) 0,05 (*) 0,05 (*) (*) 0,05 (*) 0,05 0,05 2 (**) (**) (**) (**) (**) (**) (**) (**) 0,05 (*) (*) 0,05 0,05 (5) (**) (**) 0,1 (*) 0,1 (*) (*) 0,1 0,1 (*) 0,2 0,1 (**) (*) (**) 0,1 (4) 0,5 0,02 (*)

0,02 (*) 0,02 0,02 (*) 0,02

(3) (**) (**) 0,02 (*) 0,02

0,02 (*) 0,02

(**) 0,02 (*) 0,02 (**) 0,02 (*) 0,02

2 0,02 (*) 0,02

apple, Brazilean cherry Surinam cherry (grumichama Eugenia uniflora)) and mammey sapote) sapote), (yellow canistel green sapote, other medium sized Annonaceae) Inedible peel, small peel, Inedible Inedible peel, large peel, Inedible Jambolan (java plum) (Java apple (water apple), pomerac, rose pomerac, apple), (water sapote, apple white (Java sapote, plum) (Bilimbi) (Black (java undatus)) fruit) Carambola banana) Persimmon kaki) Jambolan (Hylocereus (cactus apple Others (Virginia fruit (b) fruit pear Kiwi plantain, apple persimmon Passion dragon Prickly banana, Star Lychee (Litchi) (Pulasan, rambutan (hairy litchi), mangosteen) American or (Dwarf Others (c) Avocados pitaya Bananas Mangoes Papaya (Red Pomegranate Guava Pineapples llama and sugar apple (sweetsop), (Custard apple, Cherimoya (2) (1) 0162040 0163010 0161060 0161070 0161990 0162050 0162060 0162990 0163070 0163080 0162030 0162020 0163000 0161050 0162010 0163020 0163030 0163060 0162000 0163040 0163050 L 152/8 EN Official Journal of the European Union 11.6.2011

0,01 (*) 0,01 (10) 0,05 (*)

(9) 0,1 (*) 0,1 (8) 0,1 (*) (**) (**) (**) 0,1 (*) 0,02 (7)

0,05 (*) (*) 0,05 (*) 0,05 0,05 (**) (6)

0,05 (*) (*) 0,05 0,05 (**) (**) (**) (**) (5) (**) (**) (*) (**) 0,1 (*) 0,05 (*) (*) 0,05 (*) (*) (*) 0,05 (*) 0,05 0,05 0,05 0,05 1 0,05 (*) 1 0,05 (4) 0,02 (*) 0,02 (*) 0,02

0,02 (*) 0,02

(3) 0,1 (*) 0,1 0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02

0,02 (*) 0,02 (**) 0,02 (*) 0,02 0,02 (*) 0,02 0,1 0,05 0,1

0,02 (*) 0,02 0,02 (*) 0,02 varieties, tiger nut (Cyperus esculentus)) Tropical root and tuber vegetables tuber and root Tropical Potatoes Other root and tuber vegetables except sugar beet sugar except vegetables tuber and root Other Root and tuber vegetables tuber and Root VEGETABLES FRESH OR FROZEN OR FRESH VEGETABLES Yams (Potato bean (yam bean), Mexican yam bean) yam tannia) (Jackfruit) Mexican taro), similar fruit bean), (Japanese and (guanabana) Bread (yam Durian eddoe Soursop radish Others bean 2. (Dasheen, (i) small potatoes (a) (Potato (b) Cassava radish, Sweet Yams Arrowroot Japanese Others (c) Beetroot radish, artichokes Carrots Celeriac root (Black Jerusalem Parsnips Parsley (Angelica roots, lovage gentiana roots,) Horseradish Radishes (2) (1) 0163100 0163110 0163990 0212990 0212040 0210000 0212020 0212010 0213020 0213040 0213050 0213060 0213070 0213080 0163090 0213000 0213010 0200000 0211000 0212030 0212000 0213030 11.6.2011 EN Official Journal of the European Union L 152/9

(10)

(9)

0,1 (*) 0,1 (*) 1 (*) 0,1 0,1 1 (*) 0,1 2 (8) (*) (*) 0,02 (*) 0,02 0,04 0,04 (*) 0,02 (*) 0,02 0,02 0,02 (7) 0,04 (*) 0,05 (6) (*) 0,05 1 0,05 (*) 0,05 (*) (*) 0,05 0,05 1 (*) 0,05 0,05 (*) 1 0,05 (5) 0,05 (*) 0,05 0,05 (*) (*) (*) (*) 0,05 0,05 0,05 (*) 0,05 0,1 0,5 (*) 2 (*) 0,1 0,1 0,3 0,1 (*) 0,1 (4) 2 (+) 0,02 (*) 0,02

0,02 (*) 0,02 (3) 0,1

0,02 (*) 0,02 0,1 (*) 0,1 0,02 (*) 0,02

0,02 (*) 0,02

0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 (*) 0,02

wolfberry (Lycium barbarum and L. chinense)) Cucurbits - edible peel edible - Cucurbits Solanacea Cucurbits-inedible peel Cucurbits-inedible Fruiting vegetables Fruiting Bulb vegetables Bulb Salsify (Scorzonera, Spanish salsify (Spanish oysterplant)) (Spanish salsify varieties) gojiberry, Spanish similar Physalis, (Scorzonera, and tomato, onion Salsify onions) Swedes Turnips tree (Welsh Others (Silverskin (ii) (Pepino) Garlic onions tomatoes, Onions peppers) Shallots (patisson)) plants) Spring (Cherry fingers Others (iii) (Chilli marrow (a) (egg Tomatoes lady's Peppers squash, Aubergines Okra, Others (Summer (b) Cucumbers Gherkins Courgettes Others (c) (2) (1) 0232020 0230000 0231000 0220040 0213100 0213110 0213990 0220000 0213090 0232030 0220990 0220030 0232990 0220020 0233000 0220010 0231030 0232000 0232010 0231010 0231020 0231040 0231990 L 152/10 EN Official Journal of the European Union 11.6.2011

(10)

(9)

0,1 (*) (*) 0,1 0,1 (*) 0,1 (8) (*) 0,02 (*) 0,02 (*) 0,02 0,02 (*) 0,02 (7) 0,5 0,3 (*) 0,05 (*) 0,05 (*) 0,05 0,3 0,3 (6) 0,1 (*) (*) (*) 0,05 (*) 0,05 (*) 0,1 (*) (*) 0,1 0,1 0,1 (*) 0,1 0,05 1 (5) 0,1 (*) 0,05 (*) (*) 0,05 0,3 (*) (*) 0,05 (*) 0,1 0,1 0,05 0,3 (4)

0,1 (*) 0,1 0,02 (*) 0,02 (3)

0,02 (*) 0,02 0,02 (*) 0,02 0,1 0,1 (*) 0,1 0,5 0,02 (*) 0,02

0,1 (*) 0,1

0,02 (*) 0,02 0,1 (*) 0,1 0,02 (*) (*) 0,02 0,1 cabbage, white cabbage) (pe-tsai)) peking cabbage sum, choi (tai goo choi), flat cabbage cabbage, cow cabbage) Head brassica Head Sweet corn Sweet Kohlrabi Flowering brassica Flowering Brassicacea including plants salad other and Lettuce Leafy brassica Leafy Other fruiting vegetables fruiting Other Brassica vegetables Brassica Leaf vegetables & fresh herbs fresh & vegetables Leaf Head cabbage (Pointed head cabbage, red cabbage, savoy raab) Chinese cabbage, squash) Portuguese broccoli (Kiwano) choi, red (Winter Kale, broccoli, Melons pak cabbage, Pumpkins Portuguese Chinese Watermelons Others mustard, head (d) collards, (Calabrese, (e) (iv) (Pointed (Chinese) (a) Broccoli kale), sprouts Cauliflower cabbage (Indian Others (b) (curly Brussels Head cabbage Others (Borecole (c) Chinese Kale Others (d) (v) (a) (2) (1) 0241000 0233020 0233030 0233990 0234000 0241020 0233010 0241990 0242010 0242000 0244000 0243020 0243990 0239000 0240000 0241010 0242990 0243000 0243010 0250000 0251000 0242020 11.6.2011 EN Official Journal of the European Union L 152/11

(10)

(9) (8) (7)

(**) (**) (**) (**)

(6) (*)

0,05 (*) (**) (**) (**) (**) (5) (**) 15 3 15 (*) (**) 15 (**) 0,05 15 (*) (*) (**) 0,05 0,05 15 (4) (*) (*) 0,05 2 0,02 (*) 0,02 (*)

0,05 (*) 0,05

20 0,02 (*) 0,02

0,02 (*) 0,02 (3) 0,02 0,02 (*) 0,02

0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02

0,02 (*) 0,02 0,02 (*) 0,02

0,02 0,02 (*) 0,02 lettuce, romaine (cos) lettuce) radicchio, curld leave endive, sugar loaf) radish and up to crops (crops other harvested babyleaf brassica stage)) leaf 8 true soda)) (Salsola Agretti glassworth, sorrel, common purslane, Caraway leaves, lovage, angelica, sweet cisely and other Apiacea leaves) Spinach & similar (leaves) similar & Spinach Water cress Water Vine leaves (grape leaves) (grape leaves Vine Witloof Herbs Leaves and sprouts of Brassica spp (Mizuna, leaves of peas and peas iceberg of lettuce), chicory, leaves (cutting red-leaved (Mizuna, rosso chicory, cornsalad) spp lollo (Wild purslane, (Italian Brassica lettuce, spinach) rocket) endive) garden lettuce beetroot) of (Head amaranthus (Wild (broad-leaf lettuce), leaves, Lamb's of Lettuce sprouts cress spinach, Scarole Rucola dill (miner’s mustard Cress (Leaves and Land Zealand Rocket, leaves, purslane Red (chard) Leaves (New Coriander (Winter Others (b) leaves Spinach leaves, Purslane Beet Others (Fennel (c) (d) (e) leaves (f) Chervil Chives Celery (2) (1) 0256020 0251020 0251050 0251070 0252000 0252990 0256010 0251060 0251080 0252030 0254000 0256030 0251040 0251990 0252020 0253000 0255000 0256000 0251010 0252010 0251030 L 152/12 EN Official Journal of the European Union 11.6.2011

(10)

(9) 0,1 (*) 0,1 (*) 0,1 (8) 0,02 (*) 0,02 (*) 0,02 (7) 0,05 (*)

(**) (**) (**) (**) (**) (**) (6)

0,05 (*) 0,05 (*) (*) (*) 0,05 (*) 0,05 0,05 0,05 (**) (**) (**) (**) (**) (**) 5 5 (5) 0,1 (*) 0,2 (*) 0,1 (*) (*) 0,1 (*) 0,1 (*) 0,05 0,05 2 (*) 0,05 2 0,05 0,2 (*) 0,1 (4) (*) 0,05 (*) 0,05 2 (+) 2 (+) 0,02 (*) 0,02 0,02 (*) 0,02

(**) (**) (**) (3)

0,1 (*) 0,1 (**) (**) 0,02 (*) 0,02 (**)

0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02

0,02 (*) 0,02 0,02 (*) 0,02 0,02 (*) 0,02 0,02 2 runner bean, slicing bean, yardlong beans) bean, yardlong slicing runner bean, cowpea) Stem vegetables (fresh) vegetables Stem Legume vegetables (fresh) vegetables Legume Beans (without pods) (Broad beans, Flageolets, jack bean, lima bean, savory,) lima peppermint) summer bean, jack mint, savory, oregano) Flageolets, leaves, (Winter (laurel) beans, (Marjoram, Parsley flowers) (Balm Sage leaves Rosemary (Broad (Hyssop) Thyme (Edible Basil pods) Bay Tarragon Others (without (vi) Beans Beans (with pods) (Green bean (french beans, snap beans), scarlet Lentils snow peas)) peas, Peas (with pods) (Mangetout (sugar Others Peas (without pods) (Garden pea, green pea, chickpea) (vii) Asparagus Cardoons artichokes Celery Fennel Globe Leek Rhubarb (2) (1) 0260000 0256060 0256070 0256080 0256090 0256100 0256990 0260030 0260050 0260990 0270000 0270020 0270030 0270050 0270070 0256040 0256050 0260010 0270010 0260020 0260040 0270040 0270060 11.6.2011 EN Official Journal of the European Union L 152/13

0,01 (*) 0,01 (10) (*) 0,05

(9) 0,1 (*) 0,1 (*) (**) 0,1 (*) 0,02 (8) (*) 0,02 0,02 (*) 0,02 (*) 0,02 (*) 0,02 0,1 (*) (*) (*) (*) 0,1 (*) 0,1 (*) 0,1 0,1 0,1 0,1 0,3 (7) (*) (*) 0,05 0,05 (**) (**) (*) 0,05 (6)

0,05 (*) (*) 0,05 0,05 (*) 0,05 (*) (*) (*) (*) 0,1 (*) 0,1 (*) 0,1 0,1 0,1 0,1 (**) (**) (**) 0,1 (*) 0,1 (5) 5 (*) 0,1 (*) 0,1 (*) 0,05 (*) (*) (**) (**) 0,05 0,05 (*) (**) 0,1 (*) 0,05 (4) (*) 1 0,1 (*) 0,1

0,1 (*) 0,1 (3) 0,02 (*) 0,02 0,02 (*) 0,02 0,1 (*) 0,1 0,02 (*) 0,02 0,02 0,1 (*) 0,1

0,02 (*) 0,02 0,1 (*) 0,1 0,2

0,02 (*) 0,02 0,1 0,02 (*) 0,02

Sea weeds Sea Fungi Oilseeds beans, cowpeas) PULSES, DRY PULSES, OILFRUITS AND OILSEEDS Wild (Chanterelle, Truffle, Morel, Cep) Morel, shoots hearts Truffle, Bamboo Palm Others (Chanterelle, vetch) (viii) chickling Wild Others peas, Cultivated (Common mushroom, Oyster Shi-take) (ix) field 3. rape) (Chickpeas, Lentils Beans jack beans, lima (Broad field flageolets, beans, navy Peas turnip Lupins Others rapeseed, 4. (i) seed Linseed (Bird seed Peanuts seed Poppy seed Sesame bean Sunflower Rape Soya (2) (1) 0300030 0300040 0270090 0270990 0300000 0300020 0300010 0401010 0300990 0280000 0280990 0290000 0270080 0400000 0280010 0401020 0401030 0401040 0401050 0401060 0401070 0280020 0401000 L 152/14 EN Official Journal of the European Union 11.6.2011

0,01 (*) 0,01 (10)

(*) (*) (*) 0,02 0,02 0,02

(9) 0,1 (*) 0,1 (*) 0,05 (8) 0,02 (*) (**) (**) (**) (*) 0,02 (*) (*) 0,01 (*) 0,01 0,01 0,3 0,01 0,1 (*) (*) 0,1 0,1 (*) (**) (**) (*) (**) 0,1 (**) 0,1 0,1 (*) 0,1 0,01 (*) 0,01 (7) 0,3 0,05 10 0,05 (*) 0,02 (*) 0,02 0,1 (*) (*) 0,1 (*) 0,1 0,1 (*) 0,1 (*) (*) 0,05 (*) 0,05 0,05 (6) 0,05 (*) (*) (*) 0,05 (*) 0,05 0,05 0,05 0,1 (*) (+) (*) 0,1 2 0,1 (*) (*) (*) 0,1 0,1 0,05 (**) (**) (**) (**) (**) (**) (**) 1 (5) 1 (*) 0,05 2 2 (**) (**) (**) (**) 0,1 0,1 (*) 0,01 (*) 0,01 (*) 0,05 (*) 0,05 (4) 0,1 (*) 0,1 (3) 0,01 (*) 0,01 (**)

0,01 (*) 0,01 (**) 0,1 (*) 0,1 0,1 (*) 0,1 (**) 0,1 (*) 0,1

0,1 (*) 0,1

0,02 (*) 0,02

Oilfruits CEREALS Mustard seed seed Mustard pleasure Cotton of production Safflower kernels) Borage Pumpkin seeds (Other seeds of cucurbitacea) bean Gold oil Hempseed (palmoil Castor for Others nuts (ii) Olives Palm Palmfruit Kapok quinoa) teff) Others 5. (Amaranthus, millet, Barley Buckwheat (Foxtail Maize Millet Oats Rice Rye (2) (1) 0401120 0401130 0401140 0401990 0500020 0500060 0402020 0402030 0402040 0402990 0500000 0500010 0401080 0500030 0500040 0500050 0500070 0401090 0401100 0401110 0401150 0402000 0402010 11.6.2011 EN Official Journal of the European Union L 152/15

0,02 (*) 0,02 (10)

0,1 (*) 0,1

(9) 0,1 (*) 0,1 (8) 0,01 (*) (*) 0,01 0,01 0,05 (*) 0,05 (7) 0,05 0,1 (*) 0,1 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (6) 0,05 (*) (*) 0,05 0,05 0,05 (*) 0,05 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) 1 (5) (**) (**) (**) 0,01 (*) 0,01 (*) 0,1 (*) 0,1 (4) (**) 0,05 (*) 0,05 (**) (3) (**) (**) (**) (**)

0,1 (**) (**) (**) (**) (**) (**) (**) (**) (**)

Leaves Flowers Roots

Camellia sinensis) Herbal infusions (dried) infusions Herbal Tea (dried leaves and stalks, fermented or otherwise of Coffee beans Coffee TEA, COFFEE, HERBAL INFUSIONS AND COCOA AND INFUSIONS HERBAL COFFEE, TEA, Wheat (Spelt, triticale) nigra)) (Spelt, Sorghum Wheat Others (Sambucus 6. (i) (Elderflowers flowers (ii) flowers (iii) leaves) petals (a) flowers Camomille (linden) Hybiscus (Ginkgo Rose Jasmine leaves Lime leaves Others (b) Strawberry Rooibos root Maté root Others (c) Valerian Ginseng Others (2) (1) 0630000 0631000 0631010 0631020 0631030 0631040 0631050 0631990 0632000 0632010 0632020 0632030 0632990 0633000 0633010 0633020 0633990 0610000 0500080 0500090 0600000 0500990 0620000 L 152/16 EN Official Journal of the European Union 11.6.2011

0,02 (*) (*) 0,02 0,02 (10) 0,1 (*) 0,1 (*) 0,1 (9) 0,1 (*) 0,1 (**) (8) 0,05 (*) (*) 0,05 0,05 (7) 0,1 (*) 0,1 (**) (**) (**) (*) 0,1 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (6) 0,05 (*) 0,05 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (5) (**) (**) (**) (**) (**) (**) (**) 0,1 (*) 0,1 (**) (4) 0,05 (*) 0,05 (**) (3) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**)

0,05 (*) 0,05 Other herbal infusions herbal Other Fruits and berries and Fruits Seeds Cocoa (fermented beans) (fermented Cocoa Carob (st johns bread) johns (st Carob powder unconcentrated and hop pellets including HOPS (dried), SPICES (d) seed) (iv) (v) (Lovage 7. 8. pepper) caraway (i) seed Anise seed pink Black seed Celery seed Coriander pepper, seed Cumin pepper) Dill (Long Fennel (Japan Fenugreek Nutmeg white Others pepper (ii) and Allspice Anise berries Caraway black Cardamom Juniper Pepper, (2) (1) 0640000 0650000 0700000 0810010 0810020 0810030 0810040 0810050 0810060 0810070 0810080 0810090 0810990 0820000 0820010 0820020 0820030 0820040 0820050 0820060 0800000 0810000 0639000 11.6.2011 EN Official Journal of the European Union L 152/17

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(9) (8) (7) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (6) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (5) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (4) (3) (**) (**) (**) (**) (**) (**) (**) (**)

Bark Aril Roots or rhizome or Roots stigma Flower Buds Vanilla pods Vanilla Tamarind (Cassia) Others (iii) Cinnamon Others (iv) (Curcuma) Liquorice Ginger Turmeric Horseradish Others (v) Cloves Capers Others (vi) Saffron Others (vii) Mace Others (2) (1) 0820080 0820990 0830000 0830010 0830990 0840000 0840010 0840020 0840030 0840040 0840990 0850000 0850010 0850020 0850990 0860000 0860010 0860990 0870000 0870010 0870990 0820070 L 152/18 EN Official Journal of the European Union 11.6.2011

(*) 0,01 (*) 0,01 0,01 (10) (*)

0,05 (*) 0,05 (9) (**) (**) (**) (**) (8) 0,02 (*) (**) (*) 0,05 (7) 0,02 (*) 0,02 (*) 0,02 0,02 (*) (*) (*) 0,05 (*) 0,1 0,02 0,02 0,02 (6) 0,05 (*) 0,05 (*) 0,05 (*) 0,05 (**) (**) (**) (**) (**) (5) (**) (**) (**) 0,05 (*) (**) 0,05 (4) (*) 0,02

(3)

0,02 (*) 0,02 (**)

Bovine Swine Sheep

chilled or frozen, salted, in brine, dried or smoked or or smoked dried brine, in salted, or frozen, chilled products such processed other or meals processed as flours on these based preparations food and as sausages Meat, preparations of meat, offals, blood, animal fats fresh PRODUCTS OF ANIMAL ORIGIN-TERRESTRIAL ANIMALS ORIGIN-TERRESTRIAL ANIMAL OF PRODUCTS SUGAR PLANTS SUGAR Fat free of lean meat (root) beet 9. cane lean Sugar roots Sugar Chicory of Others 10. (i) free (a) Meat offal Fat Liver Kidney Edible Others (b) Meat offal Fat Liver Kidney Edible Others (c) Meat (2) (1) 1011050 1012050 1011030 1011990 1012990 0900020 0900030 0900990 1000000 1011040 1012040 1011010 1012010 1013010 0900010 1010000 1012000 1013000 0900000 1011000 1012030 1011020 1012020 11.6.2011 EN Official Journal of the European Union L 152/19

(10)

(9) (**) (8) (7) 0,02 (*) 0,02 (*) 0,02 0,01 (*) 0,01 (**) (**) (**) (**) (**) (**) (6) 0,05 (*) (**) (**) (**) (**) (**) (**) (**) (5) (**) (**) (**) (4) 0,05 (*) 0,05 (**) (3) (**) (**) (**)

Goat Horses, asses, mules or hinnies or mules asses, Horses, Poultry -chicken, geese, duck, turkey and Guinea fowl-, ostrich, pigeon ostrich, fowl-, Guinea and turkey duck, geese, -chicken, Poultry Edible offal Fat Liver Kidney Edible Others (d) Meat offal Fat Liver Kidney Edible Others (e) Meat offal Fat Liver Kidney Edible Others (f) Meat Fat Liver Kidney (2) (1) 1013050 1014050 1013990 1014990 1015010 1015020 1015030 1015040 1015050 1015990 1016000 1013040 1014040 1016040 1014010 1016010 1014000 1015000 1013020 1013030 1014030 1016030 1014020 1016020 L 152/20 EN Official Journal of the European Union 11.6.2011

(10)

(9) 0,05 (*) 0,05 (**) (*) 0,05 (8) 0,005 (*) 0,005 (*) 0,01 (7) 0,05 (*) 0,05 0,01 (*) 0,01 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) 0,05 (*) 0,05 (6) 0,02 (*) 0,02 0,2 (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (**) (5) (**) (**) (**) (**) (**) (**) (**) 0,05 (*) 0,05 (*) (4)

(**) (3)

(**) (**) (**)

Other farm animals (Rabbit, Kangaroo) (Rabbit, animals farm Other or sweetening matter, butter and other fats derived from milk, fats derived and other butter matter, or sweetening cheese and curd in water, or boiling cooked by steaming dried, yolks fresh, or not whether preserved or otherwise frozen moulded, sugar containing added or matter sweetening Birds' eggs, fresh preserved or cooked Shelled eggs and egg Milk and cream, not concentrated, nor containing added sugar added containing nor concentrated, not cream, and Milk Edible offal Edible Others (g) Meat offal Fat Liver Kidney Edible Others (ii) Cattle Sheep Goat Horse Others (iii) Chicken Duck Goose Quail Others (2) (1) 1030000 1020020 1016990 1020990 1017010 1017020 1017030 1017040 1017050 1017990 1020000 1030030 1030040 1030990 1020040 1017000 1030020 1016050 1020010 1030010 1020030 11.6.2011 EN Official Journal of the European Union L 152/21

(10)

(9) (**) (**) (**) (**) (8) (*) (*) (*) (*) 0,01 0,01 0,01 0,01 (7) (*) 0,05 (*) (*) 0,05 (*) 0,05 0,05 0,01 (6) (**) (**) (**) (**) (5) (**)

(4)

(**)

(**) (3) (**)

Raspberries (Wineberries, arctic (Rubus arcticus), nectar bramble/raspberry,Raspberries (Wineberries, x idaeus)) arcticus raspberries (Rubus Tomatoes (Cherry tomatoes, tree tomato, Physalis, gojiberry, wolfberry (Lycium wolfberry gojiberry, Physalis, tomato, tree tomatoes, Tomatoes (Cherry barbarum and L. chinense)) Beans (with pods) (Green bean (french beans, snap beans), scarlet runner bean, slicing bean, yardlong beans)

Other terrestrial animal products animal terrestrial Other Honey (Royal jelly, pollen) jelly, (Royal Honey Snails Amphibians and reptiles (Frog legs, crocodiles) legs, (Frog reptiles and Amphibians

Currants (red, black and white) and black (red, Currants Beans (without pods) (Broad beans, Flageolets, jack bean, lima bean, cowpea) bean, lima bean, species) jack ribes Flageolets, other beans, with fruit Pome (Broad Strawberries hybrids (iii) (b) pods) (Including Blackberries (without Gooseberries olives seed Beans Table Cotton (iv) (v) (vi) (vii) (2) (1) ) reference should be made to Annex I. MRLs apply, of plant and animal origin to the complete list of products which For a ( (*) Indicates lower limit of analytical determination analytical of limit lower (*) Indicates (R) = residue definition The differs for the following combinations pesticide-code number: (**) Pesticide-code combination for which the MRL as set in Annex III Part B applies. B Part III Annex in set as MRL the which for combination (**) Pesticide-code Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of (+) 0401090 Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of Sum of captan and folpet. captan Sum of MRL will be 2011 until 1 July valid pending submission trials of additional residue and evaluation MRL will be 2011 until 1 July valid pending submission of an additional metabolism and evaluation study’ Sum of captan and folpet. captan Sum of (+) 0152000 (+) 0153010 (+) 0154040 (+) 0231010 (+) 0260010 (+) 0260020 Ethephon (+) 0161030 1050000 1060000 1070000 (+) 0153030 (+) 0154030 1040000 Carbendazim - code 1000000: Carbendazim and thiophanate-methyl, expressed as carbendazim as expressed thiophanate-methyl, 1000000: and Carbendazim code Carbendazim - Thiofanate-methyl - code 1000000: Carbendazim and thiophanate-methyl, expressed as carbendazim Captan (+) 0130000

Journal of Pharmacognosy and Phytochemistry 2018; 7(5): 1074-1077

E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2018; 7(5): 1074-1077 Efficacy of seed treatment of fungicides, bio agents Received: 13-07-2018 Accepted: 15-08-2018 and botanicals on seed mycoflora, seed germination and seedling vigour index of mung bean RM Dolas M. Sc. Department Of Plant Pathology and Agricultural Microbiology, Post Graduate RM Dolas, SB Gawade and MC Kasture Institute, Mahatma Phule Krishi Vidyapeeth, Rahuri, Abstract Ahmednagar Maharashtra, India The seed treatment of mung bean seed with carbendazim @ 0.2 % was found most effective among all the seed treatments. It showed 8.4 per cent seed mycoflora as against 51.2 per cent in control treatment. SB Gawade The reduction in seed mycoflora due to this fungicidal treatment was 83.59 per cent over control. Among Associate Professor of of the bioagents, Trichoderma viride @ 0.6 % showed 20.0 per cent seed mycoflora as against 51.2 per cent Microbiology, MPKV, Rahuri, in control. The reduction in seed mycoflora due to this bioagent treatment was 60.93 per cent over Maharashtra, India control. Among botanicals, Garlic extract @ 0.5 % showed 21.5 per cent seed mycoflora as against 51.2 MC Kasture per cent in control. The reduction in seed mycoflora due to this botanical treatment was 58.0 per cent Associate Professor of 3Associate over control. Further, the seed treatment with carbendazim @ 0.2 % showed 87 per cent seed germination Professor of SSAC, DBSKKV, as against 69 per cent in control. The increase in seed germination due to this fungicidal treatment was Dapoli, Maharashtra, India 20.68 per cent over control. Among bioagents, Trichoderma viride @ 0.6 % showed 78 per cent seed germination as against 69 per cent in control. The increase in seed germination due to this bioagent treatment was 11.53 per cent over control. Among botanicals, Garlic extract @ 0.5 % showed 75 per cent seed germination as against 69 per cent in control. The increase in seed germination due to this botanical treatment was 8.0 per cent over control. Similarly, the seed treatment with carbendazim @ 0.2 % showed 1592.1 seedling vigour index as against 1166.1 in control. The increase in seedling vigour index due to this fungicidal treatment was 26.75 per cent over control. Among the bioagents, Trichoderma viride @ 0.6 % showed 1380.6 seedling vigour index as against 1166.1 in control. The increase in seedling vigour

index due to this bioagent treatment was 15.50 per cent over control. Among botanicals, Garlic extract @

0.5 % showed 1297.5 seedling vigour index as against 1166.1 in control. The increase in seedling vigour index due to this botanical treatment was 10.12 per cent over control.

Keywords: Triclosan, TCS, determination, detection, sensor

Introduction Green gram (Vigna radiata (L.) Wilczek) is commonly known as mung bean or mung. It is

very ancient annual crop in Indian farming. Mung bean is especially grown in Southeast Asia but some are also grown in Africa and America. In India, it is one of the most important pulse crops. It is grown in almost all parts of the country. This crop is sown usually as dry land crop in almost all the states of India, namely Madhya Pradesh, Bihar, Uttar Pradesh, Andhra Pradesh, Rajasthan, Karnataka and Maharashtra. It is an excellent source of high quality

protein and consumed in different ways. Ascorbic acid (Vitamin C) is synthesized in sprouted seeds of mung bean with increment in riboflavin and thiamine. Since mung bean is a leguminous crop, it has the capacity to fix atmospheric nitrogen through symbiotic nitrogen fixation. It is also used as green manure crop. Being a short duration crop it also provides an excellent green fodder to the animals.

Green gram is a highly nutritious containing 24 per cent of high quality protein, 1.3 per cent fats, 56.6 per cent carbohydrates and 3 per cent dietary fibers. It is rich in minerals having 140 mg calcium, 8.4 per cent iron and 280 mg phosphorous. It also contains 0.47 mg vitamin B1, 0.39 mg vitamin B2 and 2 mg niacin. It has calorific value of 334 calories per 100 g of edible protein (Baldev et al., 2003) [3].

India is the world’s largest producer as well as consumer of green gram. It produces about 1.5 to 2.0 million tons of mung bean annually from about 3 to 4 million hectares of area with an Correspondence average productivity of 500 kg per hectare. Green gram output accounts for about 10-12 % of RM Dolas total pulse production in the country. Mung production in the country remained stable more M. Sc. Department Of Plant than a decade through the 2000s at around 10 to 15 lakh tons. But a sudden jump in output was Pathology and Agricultural Microbiology, Post Graduate noted in 2010-11 to 1.75 million tonnes primarily on account of rise in production from Institute, Mahatma Phule Krishi Madhya Pradesh, Rajasthan and Tamil Nadu. In 2014-15 the mung bean production in India Vidyapeeth, Rahuri, was 1.39 million tonnes in which, Maharashtra’s contribution was about 20 %, while Ahmednagar Maharashtra, India Rajasthan was highest having 26 % of the total production. ~ 1074 ~ Journal of Pharmacognosy and Phytochemistry

Mung bean production in the country is largely concentrated Material and Method in five states viz., Rajasthan, Maharashtra, Andhra Pradesh, Mung bean seeds Gujarat and Bihar. These five states together contribute for To study the mycoflora associated with seeds of mung bean about 70 % of total Mung production in the country. There is and to test the efficacy of bio agents, botanicals and a distinct change in production pattern of mung bean across fungicides on seed mycoflora, seed germination and seedling states. Traditionally Rajasthan, Maharashtra, Andhra Pradesh vigour index, the seeds of mung bean varietiy Vaibhav were are major mung bean producing states. But there is significant collected from Pulses Improvement Project, Mahatma Phule rise in production from other states in recent years Krishi Vidyapeeth, Rahuri, Dist. Ahmednagar and Oilseed particularly, from Tamil Nadu, Uttar Pradesh and Gujarat. Research Station, Jalgaon. Nevertheless production remained volatile across the years with respect to most of the states. As per the latest available Glass wares estimates, Rajasthan, Maharashtra occupies the first two The standard corning brand glasswares viz., petriplates, positions, contributing over 45 %. Andhra Pradesh contributes conical flasks, slides and test tubes were used. about 10 % while together Gujarat and Bihar account for Equipments about 13 % of total production in the country (Anonymus, The laboratory equipments viz., autoclave, laminar flow 2015). cabinet, incubator, sterio-binocular microscope, research Seed borne diseases are regarded as major constraints in binocular microscope and weighing balance were used. mung bean production. Infected seeds serve as the source for the spread of the pathogen in disease free area. Seed infection Incubation room affects the import and export adversely because the seed The incubation room was used for keeping the blotter plates. affected with microbes is not acceptable in international The temperature of incubation room was 20 ± 20C controlled market. automatically with alternate cycle of 12 hrs. light and 12 hrs Seeds are the carrier of fungal flora either externally or darkness (Automatically controlled by electronic timer). internally. The variety and intensity of fungal flora changes area-wise and depends upon climate under which seed Miscellaneous material produced storage or in field, if, not controlled. Also they Pointed needles, inoculating needle, forceps, blotting papers, reduce seed quality i.e. seedling vigour and germination scissor, glass marking pencil, glass rods, cover slips, towel percentage. So it is necessary to control harmful fungi before papers, mercuric chloride, spirit lamp and sterilized water etc. causing damage by using suitable available measures i.e. were used. chemicals or bio agents. The seed borne pathogens associated with seeds externally or Seed treatment with bio agents, botanicals and fungicides internally may cause seed rot, seedling blight and resulting The bio agents i.e. Pseudomonas fluorescens @ 0.6 per cent into low germination. Some fungi are associated with testa and Trichoderma viride @ 0.6 per cent alone were used to and cotyledon of seeds infected in form of mycelium, find out their effect on seed mycoflora, seed germination and pycnidium and conidia or spores, after germination the seedling vigour index. Talc based formulations of these bio infection translation to hypocotyls and stem bases as well as agents were used for the seed treatment in mung bean. The dicotyledonary leaves of seedling. Some fungal seed borne weight of talc based formulations of bio agents were taken on pathogens having ability to kill the seedling or plants and weighing balance as per the dose and mixed with seeds of substantially, reduce the productive capacity (Shamsur mung bean. The material was slightly moistened with Rahman et al., 1999) [15]. Seed mycoflora play an important sterilized water, shakes slightly so as to cover the entire seed role in determining the quality and longevity of seed. surface by bio agent and then was used for blotter test and As many as 16 diseases have been reported on mung bean, seed germination. many of these diseases have been reported as seed borne. In botanical extracts, the crude extracts prepared from ginger Seedling blight, root rot, stem rot, leaf and pod rot caused by rhizome and garlic cloves were used for treatment to the Macrophomina, Curvularia, Alternaria are some major fungal mung bean seeds. The concentration of crude extract was diseases of mung bean. Species of Alternaria, Cladosporium, taken with the help of sterilized pipette and mixed with seeds Fusarium and Rhizoctonia are known to cause seed rot and so as to smear total surface of the seeds. Then this seed was pre and post-emergence losses in green gram (Khare and used for blotter test and seed germination. Chaubey, 1978; Saxena, 1986 and Patil et al., 1990) [14, 11]. In fungicidal seed treatments, required quantity of the Several workers reported Alternaria spp., Aspergillus flavus, fungicides, on the basis of their concentration was weighted Aspergillus Niger, Cladosporium spp., Colletotrichum spp., accurately. The fungicide was mixed in seed, moistened with Curvularia lunata, Fusarium spp., Macrophomina sterilized water, shake the petridish containing seeds and phaseolina, Phoma medicaginis and Rhizopus are seed borne fungicides gently so as to cover the seed surface by fungicide. and seed transmissible (Raut and Ahire, 1988; Patil et al., After drying, the seeds were used for blotter test and seed 1990) [11]. The above mentioned fungi are potentially harmful germination. for cultivation of mung bean. So it is better to use some protective measures to control these pathogens.

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Table 1: Fungicides, bioagents used and their botanicals

S. No. Fungicides, Bioagents, Botanicals Chemical name Active Ingredient Conc. Manufacturer N-(trichloromethyl thio-4) 1. Captan 50 % WP 50 % WP 0.2 % Rallis India Ltd. Mumbai Tetrachlorohexane-1-2-dicarboximide 2. Carbendazim 50 % WP 2-(Methoxy arbonyl amino) benzimidazole 50 % WP 0.2 % BASF India Ltd., Mumbai 3. Manganese ethylene bisdithiocarbamate zink Indofil Chemicals Ltd., Mancozeb 75 % WP 75 % WP 0.2 % sulphide Mumbai Carboxin (37.5 %) + Thiram (37.5 5,6- dihydro-2- Methyl-1,4- oxathiin-3- 4. 75 % WP 0.2 % Uniroyal Chemical Co. %) (Vitavax power) carboxamide (56) 5. Propineb Zinc propylenebis -dithiocarbamate 70 % WP 0.2 % Bayer India Ltd., Mumbai 6. Trichoderma viride 0.6 % 7. Pseudomonas fluorescens 0.6 % 8. Ginger extract 0.5 % 9. Garlic extract 0.5 % SVI: [Mean root length (cm) + Mean shoot length (cm)] x Seed

Germination (%) reduced the colonies of Aspergillus niger, Aspergillus flavus The per cent reduction in seed germination and seedling and Fusarium moniliforme with significantly increased seed vigour index with the inoculation of pathogens over control germination and seedling vigour index in soybean. was calculated. The data was subjected to statistical analysis Krishnamurthy et al. (2003) [8] reported that captafol and (Panse and Sukhatme, 1985) bavistin gives effective control on Macrophomina phaseolina and Fusarium spp. upto 98 %. Also Trichoderma harzianum Result and Discussion can control these fungi upto 92 % and improve seed The seed treatment of mung bean seed with carbendazim @ germination and vigour. Akhtar et al. (2005) [1] observed that 0.2 % was found most effective among all the seed in mung bean, seed treatment with Carbendazim + Carbofuran treatments. It showed 87 per cent seed germination as against was highly effective against nematode and fungal disease 69 per cent in control treatment. The increase in seed complex followed by seed powder of Azadirachta indica. germination due to this fungicidal treatment was 20.68 per Dhutraj and Gokhale (2007) [5] reported that thiram and cent over control. Among bio agents, Trichoderma viride @ bavistin was effective to control seed mycoflora of mung bean 0.6 % showed 78 per cent seed germination as against 69 per than dithane M-45 and captan. Kar and Sahu (2008) [6] noted cent in control. The increase in seed germination due to this that Trichoderma harzianum and Trichoderma viride bioagent treatment was 11.53 per cent over control. Among effectively control Macrophomina phaseolina. Also these botanicals, Garlic extract @ 0.5 % showed 75 per cent seed biocontrol agent enhanced germination and seedling vigour in germination as against 69 per cent in control. The increase in mung bean. Suryawanshi et al. (2008) [16] tested seven seed germination due to this botanical treatment was 8 per fungicides against Macrophomina phaseolina of mung bean cent over control. The seed treatment of mung bean seed with and found that carbendazim @ 0.2 % was found most effective among all the carbendazim is most effective in both field and lab condition. seed treatments. It showed 1592.1 seedling vigour index as Koche et al. (2009) [7] reported that germination percentage against 1166.1 in control treatment. The increase in seedling and seedling vigour index of soybean was increased with seed vigour index due to this fungicidal treatment was 26.75 per treatment of Thiram + Carbendazim @ 3gm/kg each and also cent over control. Among bio agents, Trichoderma viride @ reduced seed mycoflora. Chilkuri and Giri (2014) [4] studied 0.6 % showed 1380.6 seedling vigour index as against 1166.1 the seed treatment with talc based formulations of in control. The increase in seedling vigour index due to this Trichoderma viride and Pseudomonas fluorescens. These bio treatment was 15.50 per cent over control. Among botanicals, agents were tested for their efficacy against seed mycoflora Garlic extract @ 0.5 % showed 1297.5 seedling vigour index and seed germination in green gram. Among these bio agents as against 1166.1 in control. The increase in seedling vigour T. viride was found superior in controlling the seed mycoflora index due to this treatment was 10.12 per cent over control. and also maximum seed germination was observed in T. The above results on seed germination and seedling vigour viride. Chilkuri and Giri (2014) [4] reported that seed index are in confirmation with Kulshreshta (1988), Pradeep et treatment with thiram + carbendazim (2:1) @ 3 g/kg of seed al. (2000) [12], Krishnamurthy et al. (2003) [8], Akhtar et al. was increasing the seed germination, shoot length, root length (2005) [1], Dhutraj and Gokhale (2007) [5], Kar and Sahu and seedling vigour index in green gram and black gram. (2008) [6], Suryawanshi et al. (2008) [16], Koche et al. (2009) Gawade et al. (2016) studied the efficacy of bio agents, [7], Chilkuri and Giri (2014) [4], Chilkuri and Giri (2014) [4] botanicals on seed mycoflora and seed quality in mung bean and Gawade et al. (2016). Kulshreshta (1988) tested efficacy and found that Trichoderma viride @ 0.6 % + Pseudomonas of thiram, bavistin, difolaton, dithane M-45 and cereson and fluorescens @ 0.6 % was most effective among the bio agents reported bavistin as the most effective fungicide for better and Garlic extract @ 1 % among botanicals was found most emergence, less seedling mortality and better yield in mung effective in controlling seed borne pathogens and increasing bean. Pradeep et al. (2000) [12] recorded that seed treatment seed germination, seedling vigour index and field emergence with Trichoderma viride and Pseudomonas fluorescens

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Table 2: Efficacy of fungicides, bio agents and botanicals on seed germination and seedling vigour index of naturally infected seeds of mung bean (Cv. Vaibhav)

Seed Increase in seed germination Seedling vigour Increase in SVI over S. No. Treatments germination (%) over control (%) Index (SVI) control (%) 1 Captan @ 0.2 % 84 (66.50) 17.85 1503.6 22.44 2 Carbendazim @ 0.2 % 87 (68.91) 20.68 1592.1 26.75 3 Mancozeb @ 0.2 % 83 (65.66) 16.86 1477.4 21.07 4 Carboxin (37.5) + Thiram (37.5) 0.2 % 85 (67.32) 18.82 1547.0 24.62 5 Propineb @ 0.2 % 83 (65.68) 16.86 1477.4 21.07 6 T. viride @ 0.6 % 78 (62.07) 11.53 1380.6 15.5 7 P. fluorescens @ 0.6 % 76 (60.68) 9.21 1320.0 11.65 8 Ginger extract @ 0.5 % 73 (58.70) 5.47 1248.3 6.58 9 Garlic extract @ 0.5 % 75 (60.05) 8.00 1297.5 10.12 10 Control 69 (56.17) 1166.1 S. E. + 0.95 17.96

CD at 5 % 2.75 51.87 C.V. (%) 3.01 2.56

References and black gram seeds. Sustainable technology 1. Akhtar H, Ahmad V, Shukla PK. Comparative efficacy of development in crop production, 1999, 1-3. pesticides, bio-control agents and botanicals against 16. Suryawanshi AP, Gore DD, Gawade DB, Pawar AK, Fusarium oxysporum disease complex on Vigna mungo. Wadje AG. Efficacy of fungicides against Macrophomina Ann. Pl. Protec. Sci. 2005; 13(2):434-437. blight of mung bean. J Pl. Dis. Sci. 2008; 3(1):40-42. 2. Anonymous, 2015. http://www.commoditiescontrol.com/eagri-trader/ staticpages / index.php?id=89 3. Baldev B, Ramanujan S, Jain HK. Chemical composition of green gram. Pulse Crops, 2003, 363. 4. Chilkuri Ashwini, Giri GK. Detection and transmission of seed-borne mycoflora in green gram and effect of different fungicides. International Journal of Advanced Research. 2014; 5(5):1182-1186. 5. Dhutraj, Gokhale DN. Efficacy of fungicides on longevity of mung bean seeds. J Pl. Dis. Sci. 2007; 2(1):63-64. 6. Kar AK, Sahu KC. Effect of some biological agents on Macrophomina phaseolina causing seed rot, seedling blight and leaf blight of mung bean. National symposium on “plant disease scenario in organic agriculture for ecofriendly sustainablity, 2008; 10-12:59. 7. Koche MD, Kothikar RB, Anvikar DG. Effect of seed dressing fungicides and bioagents on survival of seed borne fungi and shelf life of soybean. Crop Research. 2009; 38(1-3):215-218. 8. Krishnamurthy YL, Niranjan SR, Shetty NS. Effect of chemical fungicides and biological agent on seed quality improvement in pulses. Seed Res. 2003; 31(1):121-124. 9. Kulshreshtha DO. Seed-borne infection of Fusariella hughesii in mung. Curr. Sci. 1988; 37(7):384-386. 10. Panse VG, Sukhatme PV. Statistical methods for agricultural workers, Indian Council of Agricultural Research Publication, New Delhi. 1985, 359. 11. Patil SB, Memane SA, Konde SK. Occurrence of seed- borne fungi of green gram. J Maharashtra Agric. Univ. 1990; 15(1):44-45. 12. Pradeep K, Anuja, Kanad K. Biocontrol of seed borne fungal pathogen of pigeon pea. Ann. Pl. Protec. Sci. 2000; 8(1):30-32. 13. Raut JG, Ahire SP. Seed borne fungi of green gram in Vidarbha and their control. PKV Res. J. 1988; 12(2):136- 138. 14. Saxena RM. Antagonism among seed mycoflora associated with green gram. Indian J Pl. Path. 1986; 4(2):193-194. 15. Shamsur Rahman, Suchada Vearasilpand, Sombat Srichuwong. Detection of seed borne fungi in mung bean ~ 1077 ~ 54 KAVAKA 47: 54 - 62 (2016) Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario T.S.Thind Department of PlantPathology, PunjabAgricultural University, Ludiana-141004, India Corresponding authors E.mail : (Submitted on 05-03-2016 ;Accepted on 25-05-2016) ABSTRACT Fungicides are essential component of crop protection and have played significant role in managing several devastating crop diseases. However, their indiscriminate use has resulted into development of resistance in several pathogens. This has led to poor diseasecontrol in manyinstances. The problem is more common with site-specific fungicides and performance of many of the systemic fungicides developed in the past three decades has been adversely affected. Some of the fungicide groups such as benzimidazoles, phenylamides, dicarboximides and the recently introduced strobilurins carry high resistance risk while fungicides like sterol biosynthesis inhibitors possess moderate risk. In India, development of resistance to various site-specific fungicides is now well known in some plant pathogens under practical field situations. This calls for implementation of suitable resistance management strategies to get expected disease control levels and to prolong the active life of potential fungicides. Keywords : Fungicides, resistance, plant pathogens, competitive fitness, pathogenic potential, site-specificity INTRODUCTION disease control. However, during the last two decades cases of resistance development in field situations have also been Fungicides serve as important tools for managing diseases in reported from different parts of the country (Thind, 2002; agricultural crops. Although some plant diseases may be 2008) managed through resistant varieties and alteration of cultural practices, several diseases are only managed acceptably with USE OFFUNGICIDES IN INDIA the application of a suitable fungicide. About 150 different The use of fungicides in India is quite low as compared to chemicals belonging to different classes are used as developed countries. Overall fungicide use is less than fungicides in various countries including India. insecticides and herbicides. The consumption of fungicides in Resistance to fungicides has become a challenging problem 2009 was 8307 MT compared to 26756 MT of insecticides in the management of crop diseases and has threatened the and 6040 MT of herbicides with market share at 19% performance of some highly potent commercial fungicides compared to 61% of insecticides and 17 % of herbicides. The (Brent, 1995). Unlike insecticides, where resistance problems current fungicide market in India is worth Rs. 4300 millions. are known to occur much earlier, practical problems of Apart from conventional compounds like sulphur, fungicide resistance has emerged much later in 1970's and dithiocarbamates, copper-based, mercurials, phthalimides, thereafter. Worldwide, resistance in pathogen populations to etc., several of the site-specific fungicides of the groups like more than 100 different active ingredients has been reported. benzimidazoles, oxathiins, thiophanates, organophosphorus, Incidence of resistance to fungicides has remained restricted triazoles and related sterol inhibitors, phenylamides, mainly to systemic fungicides that operate against single bio- strobilurins and other recently developed compounds are chemical targets also known as single site inhibitors (Dekker, being used in India for controlling different diseases on a 1985; Brent, 1995). These site-specific systemic fungicides number of crops. As many as 52 fungicides belonging to were introduced in the mid 1960's onwards and include different groups were registered for use in India (Table 1 ). In several major groups of fungicides such as benzimidazoles, addition, formulations of combination products containing pyrimidines, phenylamides, sterol biosynthesis inhibitors, systemic and contact fungicides are also registered. dicarboximides, phenylamides, etc.. During the past decade, more novel compounds with different modes of action Crop wise consumption of fungicides in India is maximum on notably phenylpyrroles, anilinopyrimidines, strobilurins, pome fruits (12.7%), closely followed by potatoes (12.2%), spiroxamines, phenylpyridylanines, quinolines, etc. have rice (12.0%), tea (9.4%), coffee (8.0%), chillies (7.6%), been developed having bioefficacy against diverse plant grapevines (6.9%), other fruits (5.9%), other vegetables diseases. Several of these modern selective fungicides have (4.6%) and other crops which account for about 75% of the become vulnerable to the risk of resistance development in total fungicides used in India (Thind, 2002). target pathogens in different countries (Brent and Hollomon, ACQUIRED RESISTANCE IN LABORATORY 2007) In India, several cases of adaptive resistance to many As compared to developed countries, not much work has been fungicides including multisite-action compounds have been done on the problem of fungicide resistance in India. This reported under laboratory conditions by different workers, could be attributed to the lack of awareness among the Indian but their possible implications in disease control have not workers about the importance of the problem and non- been indicated. Various methods such as adaptation to availability of trained scientific manpower in this field. Most increasing fungicide concentrations, exposure to UV of the earlier studies done on fungicide resistance in India radiations and chemical mutagens have been employed to pertained to acquired resistance using mutagens or training study resistance development in diverse fungi to (pressurization) methods under laboratory conditions, dithiocarbamates, copper-based, oxathiins, benzimidazoles, without looking into their possible implications in practical T.S. Thind 55 orchards in Kashmir valley, sensitivity studies of conidial Table 1. Site-specific fungicides registered for use against populations ofV. inaequalis from 40 affected orchards were carried out. Apparently one isolate was obtained from each Fungicide group Name of the fungicide orchard. The results were reported to indicate mancozeb Oxathiins Carboxin, Oxycarboxin resistant strains in 12 orchards and carbendazim resistant Benzimidazoles Benomyl, Carbendazim Guanidines Dodine strains in 3 orchards (Basu Chaudhary and Puttoo, 1984). Thiophanates Thiophanate methyl Phosphorothiolates Edifenphos, Iprobenfos Although apparently unusual, mancozeb resistant isolates Dicarboximides Iprodione could tolerate 2.5 times higher levels of the fungicide Acylalanines Metalaxyl, Metalaxyl-M (Mefenoxam) compared to sensitive isolates and the per cent disease control Cyano-acetamide oximes Cymoxanil ranged from 41 to 77 in these orchards. The isolates proved Cinnamic acid derivatives Dimethomorph pathogenic on young apple foliage of cv. Red Delicious Trizoles Propiconazole, Penconazole, Myclobutanil, Triadimefon, during the first and second sub-culturings only.The resistance Bitertanol, Hexaconazole, was, however, found to be unstable as these isolates lost the Difenoconazole, Tebuconazole, Flusilazole character after three sub-culturings on mancozeb free Morpholines Tridemorph medium and became non-sporulating. Since mancozeb Pyrimidines Fenarimol Melanin biosynthesis Tricyclazole, Carpropamid resistance in these isolates was not found to be stable by the Inhibitors workers themselves and in any case was at a relatively low Dithiolanes Isoprothiolane level, the reduction in disease control over some years could Stobilurins Azoxystrobin, Kresoxim methyl, Trifloxystrobin possibly be attributed to the poorly managed spray schedules Oxazolidinones Famoxadone of mancozeb. This fungicide stands low risk of resistance Imidazoles Fenamidone Valinamides Iprovalicarb development in the pathogens due to its multi-site mode of Anilides Thifluzamide action, and cases of practical resistance to mancozeb have not Antifungal antibiotics Aureofungin, Kasugamycin, Validamycin been reported elsewhere, despite its widespread use against Source : Central Insecticides Board (www.cibrc.nic.in) many pathogens. organophosphorus, phenylamides, alkyl phosphonates, The carbendazim resistant isolates could tolerate 3-14 times morpholines and antifungal antibiotics in the laboratory higher levels of the fungicide. These are relatively low (Thind, 1995). resistance factors compared with carbendazim resistance reported elsewhere. In contrast to the mancozeb resistant RESISTANCE DEVELOPMENT IN FIELD isolates, the carbendazim resistance was found to be stable. The increase in use of fungicides, particularly of selective The isolates retained the spore producing character and were fungicides, on important crops caught the attention of some pathogenic on young apple foliage during all sub-culture workers about their likely effects on pathogen populations Table 2. Reported cases of fungicide resistance under field and in the past years, cases of fungicide resistance situations in India development have also been reported under field conditions in India (Thind, 2002; 2008). Sensitivity studies through Fungicide Pathogen (Host) Reference regular monitoring of conidial/sporangial populations of Carbendazim Venturia inaequalis Basuchaudhary and several pathogens have led to the detection of fungicide (Apple) Putto (1984) resistant strains with low to high resistance levels in some Gloeosporium Kumar and Thind ampelophagum (Grapes) (1992) plant pathogens. Some reported field cases of fungicide Aspergillus flavus Gangawane and resistance in India are mentioned inTable 2 and are (Groundnut) Reddy (1985) described in the following pages. Cercospora beticola Pal and Benzimidazoles (Sugarbeet) Mukhopadhyay (1983) Apple scab (Venturia inaequalis ) : Apple scab caused by V. Edifenphos Dreschlera oryzae (Rice) Annamalai and Inaequalis (Cke.) Wint. has become endemic in all the Lalithakumari (1990) important apple growing belts covering an area of 60, 000 ha Pyricularia oryzae (Rice) Lalithakumari and Kumari (1987) in Kashmir alone. As most of the commercial apple cultivars Metalaxyl Plasmopara viticola Rao and Reddy (1988) are susceptible to scab, orchardists mainly depend on (Grape) fungicides such as mancozeb, zineb, ziram, carbendazim, Phytophthora infestans Arora et al.(1992) benomyl, captan, triazoles, etc. for its control. At least six (Potato) applications of fungicides are recommended against this Thind et al.(1999) disease in Kashmir and seven in Himachal Pradesh (Gupta Phytophthora parasitica Thind et al.(2009) and Gupta, 1996) at various phenological stages starting from (Citrus) silver tip/green tip stage till harvest. But the growers usually Pseudoperonospora Thind et al.(2011) give 12 -15 applications of different fungicides to ensure good cubensis (Cucumber) disease control. Based on the observations by some growers Oxadixyl Phytophthora infestans Singh et al.(1993) on decreased level of disease control after prolonged and (Potato) exclusive usage of mancozeb and carbendazim in their Triadimefon Uncinula necator Thind et al.(1998) (Grape) 56 Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario inoculations. Strategies for the management of fungicide were categorised into three morphological groups and resistance inV. inaequalis involving need-based application majority of the resistant isolates produced reddish brown to of fungicides and the use of sanitary, physical and cultural peach red colonies (Thindet al ., 1994). practices to control the pathogen multiplication have been Further studies conducted from 2000-2004 revealed that suggested by Putto and Basu Chaudhary (1986). carbendazim resistant isolates ofG. ampelophagum were Grape anthracnose (Gloeosporium ampelophagum ): persistent in natural populations and could be detected Anthracnose, caused byG. ampelophagum (de Bary) Sacc. frequently in the vineyards around Ludhiana in Punjab state (syn.Elsinoe ampelina ), poses a serious threat to grape (Mohanetal ., 2005). cultivation in Punjab and other parts of India and requires Pathogenic behaviour of resistant isolates: Pathogenic regular fungicide applications for its control. A number of behaviour of two resistant and two sensitive isolates was treatments of benzimidazole and related fungicides like studied on detached leaves of cv. Perlette treated with carbendazim, benomyl and thiophanate methyl, as well as different concentrations of Bavistin in the laboratory. While conventional contact fungicides (copper-based, the sensitive isolates did not produce any symptoms above dithiocarbamates, phthalimides, etc.) are applied repeatedly 250 g/ml, both the resistant isolates Ga 28 and Ga 53 by the growers to protect the plants from this disease. Due to developed normal sporulating lesions at 500 g/ml and also excessive and irrational use of benzimidazoles, development produced mild symptoms even at 1000 g/ml of Bavistin thus of resistance, associated with inferior disease control, has confirming their resistant character. been observed inGloeosporium ampelophagum and the strains with high level of resistance to carbendazim have been Cross resistance to other fungicides: Cross resistance to isolated from vineyards in the Punjab state (Kumar and other fungicides viz. Topsin-M (thiophanate methyl) , Captaf Thind, 1992). Studies were conducted during 1990-97 to (captan), Indofil M-45 (mancozeb), Bordeaux mixture determine the population structure ofG. ampelophagum with (copper sulphate + calcium hydroxide), and Bayleton- 5 regard to fungicide sensitivity and strategies for its (triadimefon) was studied by growth inhibition assay as well management. as by detached leaf assay by taking one resistant and one sensitive isolate. Observations revealed that resistant isolate Screening for carbendazim resistance : In the preliminary Ga 53 possessed cross resistance to Topsin-M which has a screening a total of 80 isolates ofG. ampelophagum collected similar mode of action as Bavistin (Mohan and Thind, 1995). from various regions in the Punjab state during 1990-97 were On the other hand both resistant and sensitive isolates studied for their sensitivity to carbendazim (Bavistin 50 WP) exhibited sensitive response to all other fungicides tested. In using malt agar plates amended with 1 and 5 g/ml of another study ( Thindet al ., 1997) on cross resistance, three carbendazim. Majority of the isolates showed sensitive or triazole fungicides viz. Score (difenconazol), Corail weakly resistant response and were unable to grow beyond 1 (tebuconazol) and Olymp (flusilazole) and one pyridylanine g/ml of carbendazim. However, 36 % of the isolates showed compound Dirango (fluazinam) were found to possess high growth at 5 g/ml indicating resistant response to carbendazim inhibitory action against carbendazim resistant as well as (Thind and Mohan, 1998). Twenty three isolates found sensitive isolates with MIC values of triazoles ranging resistant in the preliminary screening were further grown at between 1-5 g/ml for both the types. Fluazinam also exhibited higher concentration up to 100 g/ml of carbendazim. Of these, good efficacy at 10 g/ml and above. By detached leaf assay all the isolates showed normal growth up to 50 g/ml while 15 difenconazole proved most effective and no symptoms were able to grow even at a higher dose of 100 g/ml of developed at 25 g/ml. Fluazinam arrested diseases carbendazim thus exhibiting high resistance factors (Table development completely at 500 g/ml by both the isolates and 3). These isolates were obtained from vineyards receiving holds promise alongwith difenconazole to check resistance. regular treatments of Bavistin and were mostly from areas near Ludhiana. Resistance to carbendazim was found to be Management of carbendazim resistance: Indofil M-45 and persistent in nature as the resistant isolates were able to grow Bordeaux mixture to which carbendazim resistant isolates did at 50 g/ml of carbendazim even after one year of sub-culturing not show any cross resistance were tested in a resistance on fungicide-free medium. The isolates of G. ampelophagum affected vineyard near Ludhiana. Bavistin (0.1%) when used alone did not provide desired control of grape anthracnose. In Table 3. Structuring ofGloeosporium ampelophagum isolates contrast, when it was applied in alternation with Bordeaux from different vineyards for carbendazim sensitivity in mixture (2:2:250) or Indofil M-45 (0.3%) there was Punjab (1990-1997) significant reduction in disease severity (Mohan and Thind, Number of ED50 MIC Resistance Sensitivity 1995). Triazole fungicides such as difenconazole, Isolates tebuconazole, flusilazole and fluazinam, a pyridylanine (?g /ml) (?g/ml) factor class tested compound, which showed promising efficacy against 36 0.02-0.04 0.05-0.1 0.0 S resistant and sensitive isolates ofG. ampelophagum in 21 0.04-0.16 0.20-0.5 1.8-4.5 WR laboratory studies using detached leaf assays (Thindet al. , 1997; Thind and Mohan, 1998) are now used in field 8 14-50 40-100 360-900 HR conditions as anti-resistance measures. 15 69-100 > 100 > 900 HR S = Sensitive, WR = Weekly resistant, HR = Highly resistant Application of an effective fungicide immediately after first Source : Thind and Mohan (1998) rain shower in March/April helps in checking the primary T.S. Thind 57 infection and multiplication of inoculum for subsequent Table 4. Sensitivity range ofDrechslera oryzae field isolates infections, thereby, reducing fungicide sprays and against edifenphos minimising the risk of resistance development to site-specific fungicides like carbendazim. ED50 Range Per cent of isolates (mg/ml) 1984 1985 1986 1987 1988 Sugarbeet leaf spot (Cercospora beticola ) : Leaf spot of sugarbeet, caused byC. beticola Sacc. is a serious disease 20 - 50 96 74 64 50 48 problem of sugarbeet in India. Various fungicides including 51 - 100 4 26 16 18 16 carbendazim formulations are widely used to control this 101 - 150 0 0 20 18 10 disease. Some natural populations of the fungus were 151 - 180 0 0 0 14 26 screened for sensitivity to carbendazim (Bavistin 50 WP). It Source : Annamalai and Lalithakumari (1990) was surprising to note that a natural mutant from untreated strain and exhibited ED value of 45-50 M. Cross resistance field was able to tolerate high concentrations of carbendazim 50 (Paland Mukhopadhyay, 1983). was observed to iprobenphos, an organophosphorus fungicide with same mode of action. Other fungicides tested such as Organophosphorus Compounds copper oxychloride, benomyl, bitertanol, carbendazim and Brown spot of rice (Drechslera oryzae ) : Organophosphorus pyroquilon were less effective against resistant isolates and fungicides such as edifenphos and iprobenphos are widely inhibited the growth of the fungus at higher concentrations used in southern parts of India for the control of brown leaf (Annamalai and Lalithakumari, 1992). Cross resistance spot of rice caused byD. oryzae (Breda de Haan) Subram. and studies and field treatments indicated that edifenphos Jain, which causes severe crop losses if not controlled in early resistance inD. oryzae can be counteracted by spraying stages. Edifenphos (Hinosan) is used quite regularly in Tamil mancozeb as an alternative (replacement) fungicide. Nadu and other rice growing states for reducing crop losses However, edifenphos is still used againstD. oryzae and is due to this disease. Risk of resistance development to working well in most of the areas. edifenphos has been determined inD. oryzae under selection Blast of rice (Pyricularia oryzae) : Blast disease of rice pressure of the fungicide in field during 1984 -1988 at a caused byPyricularia oryzae Cav. (syn. P. grisea Cav. and village farm near Chingleput, Madras (Annamalai and Magnaporthe oryzae ) is a serious disease in rice - growing Lalithakumari, 1990). Field isolates of the pathogen were areas of South Indian states. Edifenphos (Hinosan) is widely collected to study the base-line data on the sensitivity before used in foliar applications for the effective control of this commencing the application of edifenphos in 1984 and disease. A preliminary study has been done to estimate the subsequently after the application of edifenphos every year sensitivity of natural populations ofP. oryzae isolates to up to 1988. Repeated applications of edifenphos resulted in edifenphos (Lalithakumari and Kumari, 1987). Diseased patches of paddy crop cv. IR-50 with severe disease leaves of rice cv. IR 50 were collected from fields sprayed manifestation. The disease intensity in the treated plots was regularly with edifenphos and fifty monosporic isolates of surprisingly much higher than in untreated plots. Every year the pathogen were obtained. Sensitivity of these isolates to 400 leaf samples were collected at random, the pathogen was edifenphos was tested at ten concentrations ranging from 10 isolated (one lesion/plate) and screened for edifenphos to 100 m on oat meal agar by mycelial growth inhibition sensitivity by measuring the radial growth on PDA amended technique. The ED50 values were compared with a sensitive with 10, 20, 50, 200 and 300 g/ml of the fungicide. To isolate, unexposed to edifenphos. characterise the isolates for resistance, these were grouped The sensitive isolate had ED value of 36.3 M. Seventeen into four categories based on ED50 values i.e., sensitive, low 50 level resistance, moderate resistance and high level resistance isolates from treated fields showed a shift in their ED50 values having ED50 below 50, between 50-100, between 101-150 and above 50 M and out of these 17 isolates, 9 isolates had ED50 above 150 g/ml, respectively. values above 60 M (up to 75.8 M) thus indicating resistant response to edifenphos. Not much variation was observed in A shift in the level of sensitivity to edifenphos was noticed the growth pattern, conidial morphology and pathogenicity from year to year. In 1984, before the application of among the fifty isolates tested. The resistant isolates were edifenphos, 96 per cent of the isolates were sensitive to equally pathogenic when inoculated on one month old edifenphos at 50 g/ml, while in 1985, 1986, 1987 and 1988 seedlings of IR 50 rice cultivar. In cross resistance studies to (i.e. after fungicide application), 74, 64, 50 and 48 per cent other fungicides the resistant isolates showed positive cross respectively of isolates showed the same level of sensitivity. resistance to iprobenphos, a related fungicide. Ziram The sensitivity data of the field isolates thus showed a clear effectively inhibited thegrowth of all the resistantisolates and shift in the level of sensitivity ofD. oryzae due to frequent its use was suggested as a companion fungicide in mixture applications of edifenphos (Table 4 ). Rate of uptake of with edifenphos or as alternate spray fungicide edifenphos was less in resistant strains ofD. oryzae and the (Lalithakumari and Mathivanan, 1990). reduced membrane permeability was suggested as the mechanism of resistance. Phenylamides When tested for cross resistance to other fungicides, Grape downy mildew (Plasmopara viticola ) : Grape downy mancozeb showed significant inhibitory effect on the growth mildew caused byP. viticola (Burk. & Curt.) Verl. & de Toni of edifenphos resistant isolates compared with the sensitive causes severe losses in southern states of India such as 58 Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario Maharashtra, Andhra pradesh and Karnataka, especially on Potato late blight (Phytophthora infestans ) : For the two commercial grape varieties Anab-e-Shahi and Thompson management of late blight of potato caused by P. infestans Seedless affecting the production and quality of grapes. When (Mont.) de Bary, traditional fungicides such as mancozeb, metalaxyl became available in late 1970s, it caught the zineb, copper oxychloride, chlorothalonil, etc. have been in attention of Indian farmers who found it miraculous in use since many years in India. However, these fungicides controlling grape downy mildew which was earlier difficult to provided poor disease control under heavy disease pressure. be controlled by the traditional contact fungicides. The introduction of phenylamide fungicides provided much needed relief to the Indian farmers as these provided excellent The grape growers around Hyderabad started using metalaxyl control of late blight even under severe disease conditions. (Ridomil 25WP) in 1981 to control downy mildew. Based on Metalaxyl in combination with mancozeb (Ridomil MZ reports by some grape growers in 1986 regarding the loss of 72WP) was commercially introduced in India during autumn effectiveness of metalaxyl in controlling grapevine downy 1988 and since then is being widely used for the control of late mildew in areas around Hyderabad, monitoring and blight in different potato growing areas of the country. Now sensitivity studies were undertaken for ascertaining the cause Ridomil Gold having metalaxyl-M (also called mefenoxam) for reduced efficacy of metalaxyl (Rao and Reddy, 1988). has been introduced recently in India for control of late blight. Infected leaves were collected from three affected orchards Although the mixture fungicides are expected to delay the situated at three villages near Hyderabad which had received onset of resistance build up, their use does not guarantee metalaxyl applications for 3, 4 and 3 years, respectively. prevention of resistance development (Gisi and Staehle- Sporangial populations from these samples were assayed for Csech, 1989). sensitivity to metalaxyl (Ridomil 25 WP) at 25, 50, 100 and 250 g a.i./ml following detached leaf method of Pappas Following the reports of resistance development to metalaxyl (1980). The preliminary sensitivity assays with P. viticola in other countries ( Davidseet al ., 1981; Davidse, 1987), populations indicated that the loss of efficacy of metalaxyl in monitoring for metalaxyl resistant strains ofP. infestans was these vineyards was attributed to the development of carried out in Nilgiri hills from 1989-91 (Aroraet al., 1992). resistance to metalaxyl which had been used frequently by the Sporangial populations ofP. infestans collected from potato growers (Rao and Reddy, 1988). Considerable difference in fields sprayed with Ridomil MZ were analysed for their minimal inhibitory concentration was seen among the three response to metalaxyl by detached leaf method. Metalaxyl populations. resistant isolates of the pathogen were absent from early to mid summer potato crop seasons. These, however appeared Metalaxyl was almost completely inactive against pathogen towards the end of summer season starting from last week of populations collected from two of the three villages which July and a maximum frequency of 13% in the autumn. confirmed the reports of grape growers regarding the loss of Variations in tolerance to metalaxyl from 50 to 700 g/ml were efficacy of metalaxyl in controlling grape downy mildew observed among different isolates resistant to metalaxyl. (Table 5 ). The continuous and exclusive use of metalaxyl (Ridomil 25 WP) by grape growers at these villages for 3-4 The highly tolerant isolates (300 to 900 g/ml) were observed years had led to the development of resistant populations of P. only during the autumn season and comprised up to 6% of the viticola. No further work has been done on this problem after total samples examined. The resistant isolates could be this report. Several rounds of metalaxyl based combination obtained in plots with a combined spray of metalaxyl and products with mancozeb viz. Ridomil-MZ (now Ridomil mancozeb and also in plots with individual sprays of Gold) are used for the control of grape downy mildew in mancozeb or chlorothalonil and the control plots later during India. Although these combination products are known to the season. The resistant isolates were found to be more minimise the risk of resistance development, regular aggressive in traits like short incubation period, quick monitoring for determining the changes in sensitivity levels germination of sporangia to zoospores, and ability to cause of pathogen populations is necessary where these fungicides larger lesions, as compared to the sensitive isolates (Arora, are used frequently. A simple laboratory technique based on 1994). In another study of resistant monitoring in Nilgiri Hills sporulation on leaf discs has been developed for laboratory following leaf disc assay, Gangawaneet al. (1995) have testing of fungicides (Thindet al ., 1988) which requires less reported that out of isolates ofP. infestans tested, 82% were space and can be easily employed for determining fungicide sensitive, 5% were moderately resistant ( RF 15-40) and 4% resistance in a large number ofP.viticola populations. were highly resistant ( RF 60-70). Use of metalaxyl in mixture with chlorothalonil was highly effective against both Table 5. Sensitivity ofPlasmopara viticola populations to sensitive and metalaxyl resistant isolates. Resistance to metalaxyl by detached leaf assay metalaxyl has also been reported inP. infestans from Shimla ED50 Range Per cent of isolates hills after 5 years of use of Ridomil MZ by Singhet al . (1993) (mg/ml) 1984 1985 1986 1987 1988 but the resistance level reported is quite low. They have also reported isolates of this pathogen developing moderate 20 - 50 96 74 64 50 48 resistanceto oxadixyl under experimental conditions. 51 - 100 4 26 16 18 16 In the Punjab state of India, metalaxyl in mixture with 101 - 150 0 0 20 18 10 mancozeb (Ridomil MZ, Matco 8-64) is being widely used 151 - 180 0 0 0 14 26 for the control of late blight of potato since 1989. Quite often, Source : Annamalai and Lalithakumari (1990) farmers also use self-prepared mixtures (tank-mixed) of T.S. Thind 59 metalaxyl (35% SD) and mancozeb in various proportions. Sensitivity levels of 68P. infestans populations collected during 1996-1999 crop seasons from various fields treated M RS with fungicides in Punjab were monitored for their sensitivity to metalaxyl following detached leaf method (Thind et al.,1989). Thirty one populations, mostly from Hoshiarpur district, showed mild to severe infection at 10 g/ml, while 12 populations, collected mostly during 1998-99 showed varying levels of infection at 50 g/ml in the initial screening. When tested at higher concentraions of metalaxyl, three populations were able to produce symptoms at 100 and 200 g/ml thus showing higher resistant response to metalaxyl (Thindet al., 2001). The resistance factors of populations with varying levels of decreased sensitivity to metalaxyl ranged between 2.8 to 28.5.Amarked decrease in the efficacy of Ridomil MZ was also observed in the field from where Fig. 1 Metalaxyl resistant and sensitive strains of highly resistant populations were collected. Phytophthora infestans showing amplification with P9 During 2005-2008 crop seasons, 48 sporangial populations Source: Kauret al .(2010) ofP. infestans collected from different potato growing areas metalaxyl sensitive or tolerant strains ofP. infestans (Cohen in Punjab were tested for metalaxyl sensitivity among these et al., 1995). Metalaxyl resistance was effectively managed 10 populations showed resistant response causing infection at under field conditions through application of novel action 200 µg/ml of metalaxyl with resistance factor up to 60 (Kaur fungicides such as Infinito 68.75 SC (fluopicolide+ et al., 2010). The resistant population exihibited competitive propamocarrb chloride), Amistar 25 SC (azoxystrobin), fitness in a mixture with sensitive population (Table 6 ). Acrobat 50 WP (dimethomorph), Mandipropamid 250 SC RAPD analysis of metalaxyl resistant populations of and Curzate M-8 72 WP (cymoxanil + mancozeb). P.infestans was done with 10 oligonucleotide primers. Of 50 Combination of fungicides with different modes of action primers initially used for amplification, 23 showed retards development of resistance and ensure sustainable Table 6. Competitive fitness of metalaxyl resistant and management of late blight. The potential of several of these sensitive populations of Phytophthora infestans new fungicides has been documented in a recent review (Stevenson, 2009). Metalaxyl PDI with different combinations of R and S populations concs. (mg/ml) R S R (50):S (50) R (25): S (75) R (75): S (25) Cucumber downy mildew (Pseudoperonospora cubensis ) : 0 86.0 85.0 74.8 74.1 75.6 10 65.3 13.4 38.5 29.3 71.1 Phenylamide fungicides are regularly used by farmers in 50 49.2 0.0 31.2 23.3 46.6 various states of India including Punjab to manage downy 100 44.2 0.0 22.1 7.2 33.5 mildew infection on cucurbits such as cucumber and melons. R = Resistant strain, PI-24; S = Sensitive strain, PI-31; PDI=Per cent disease index Source : Kaur et al. (2010) Sensitivity changes to metalaxyl in Pseudoperonospora cubensis populations collected from cucumber and polymorphism and 10 were able to distinguish resistant and muskmelon fields were monitored during 2007 and 2008. susceptible populations producing 2-3 unique bands. Maximum number of sporangial populations (12 out of 25) Information on banding pattern for all the primers was used to exhibited resistant response were collected in district determine genetic distance between resistant and sensitive Amritsar with ED90 values of metalaxyl ranging between 30- isolates and to construct a dendrogram. RAPD data 150 g/ml and resistance factor (RF) between 6-30. Most of the distinguished the test isolates into two groups thus separating populations from Jalandhar and Kapurthala showed normal the resistant and susceptible isolates. Using primer P 9 a sensitive response. Resistant populations possessed normal unique band of 100bp was found in susceptible (S) isolates pathogenic potential and exhibited strong competitive fitness indicating that these isolates are different from resistant ones when inoculated in mixture with sensitive populations (Table in this 100bp region (Fig.1 ). 7). Resistant populations did not show cross resistance to fungicides with different modes of action such as The resistant populations were found to be highly pathogenic azoxystrobin, cymoxanil, dimethomorph, fluopicolide, when inoculated on potato leaves of cv. Kufri Chandramukhi. propamocarb, chlorothalonil and mancozeb. These Their disease severities (78 - 90 per cent) were comparable fungicides were also found effective against metalaxyl with those of sensitive populations (82 - 100 per cent). resistant populations under field condition and can form a part Incubation period varied from 4 to 5 days in both resistant and of the strategy to manage metalaxyl resistance in practice sensitive populations. Sporulation was also comparable in (Thindet al .,2011) both types of populations. No cross resistance was observed to dimethomorp, mandipropamid, cymoxanil, benalaxyl, Citrus foot rot (Phytophthora parasitica ): Metalaxyl based previcur, fluopicolide, azoxystrobin and multisite contact fungicides are commonly used to manage foot rot of citrus (P. fungicides chlorothalonil, fluazinam and mancozeb. parasitica) in different states of India. A significant reduction Dimethomorph has been reported to be effective in in fungicide efficacy has been observed in many orchards controlling late blight of potato and tomato caused either by over the years. In Punjab state, investigations were carried out 60 Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario and Mohan, 1995). Majority of the populations showed Table 7. Pathogenic potential of metalaxyl resistant (R) and typical sensitive reaction as their germtubes measured less sensitive (S) population of Pseudoperonospora cubensis than 250 at 0.3 g/ml which was taken as the discriminatory concentration to distinguish resistant strains in the conidial P.cubensis MIC Incubation Disease Sporulation population (mg/ml) period severity (spores/cm2) populations. Germtubes of such conidia were distorted and (Days) (%) deformed at the tips and comparable in their response with the DM-12 (R) 100 5 80 75.5 × 103 reference sensitive strainAne-17. However, three populations 3 DM-13 (R) 150 4 85 73.0 × 10 showed 4 to 6 per cent conidia with normal germtubes DM-15 (R) 10 4 90 75.0 × 103 DM-16 (R) 10 4 80 72.0 × 103 measuring more than 250 at a higher concentration of 3 g/ml P.cubensis MIC Incubation Disease Sporulation of triadimefon. Two of these populations, 1a from Bangalore population (mg/ml) period severity (spores/cm2) and 7a from Pune, showed one per cent conidia producing (Days) (%) normal germtubes at a still higher concentration of 10 g/ml 3 DM-12 (R) 100 5 80 75.5 × 10 thus demonstrating low to moderate levels of resistance DM-13 (R) 150 4 85 73.0 × 103 DM-15 (R) 10 4 90 75.0 × 103 development in these populations (Thindet al ., 1998). These DM-16 (R) 10 4 80 72.0 × 103 three populations also developed sporulating colonies on the R=Resistant population, S= Sensitive population leaf discs at 3 and 10 g/ml thus confirming resistance to Source : Thind et al. (2011) triadimefon. However, no apparent decline in the field performance of this fungicide was observed except in the to determine changes in sensitivity levels of P. parasitica vineyard at Bangalore where a reduced disease control was isolates from different citrus orchards where reduced efficacy recorded. have been reported after metalaxyl applications. Of the 56 isolates of the fungus tested, 9 isolates showed resistant Cross resistance to other SBI fungicides : One isolate each response with ED50 values of metalaxyl ranging between 38- from populations 1a and 7a, showing moderate resistance to 200 g/ml. Pathogenic potential, colony growth and triadimefon, was further studied for sensitivity to two other sporulation of the resistant isolates were comparable with sterol inhibiting fungicides, triadimenol (Baytan 5) and sensitive isolates (Thindet al ., 2009). The resistant isolates fenarimol (Rubigan 4) by sporulation test on treated leaf did not show cross resistance to azoxystrobin, cymoxanil, discs. Compared to the sensitive strainAne-17, which showed fluopicolide and previcur. Cymoxanil is providing effective negligible sporulation at 0.3 g/ml of triadimenol, isolate 1a control of foot rot in the orchards where metalaxyl resistance and 7a produced some sporulation even at 1 g/ml thus has been a problem. A leaf disc assay involving fungicide confirming cross resistance to triadimefon. However, on treated leaf discs of rough lemon rootstock placed on soil fenarimol treated discs, the two isolates were found to be slurry has been developed for early detection of metalaxyl equally sensitive as the reference strain, thereby, showing no resistance in citrus orchards (Thindet al. , 2015). cross resistance to fenarimol (Thindet al ., 1998). Fenarimol is now being used where reduced sensitivity has been Triazoles observed to triazolefungicides. Grape powdery mildew (Uncinula necator ) : Powdery Triazoles and other sterol inhibiting fungicides such as mildew, incited byU. necator (Schw.) Burr., is another bitertanol (Baycor), hexaconazole (Contaf), myclobutanil serious disease of grapevine in India causing more damage to (Systhane), penconazole(Topas) and fenarimol (Rubigan) are the developing berries. For the past 20 years, various DMI also being used at present for controlling apple scab in India. fungicides, in addition to the traditional sulphur and dinocap, Reduced sensitivity to these fungicides in this pathogen has are being used for controlling this disease. Apart from not yet been reported from Indian orchards Since Venturia triadimefon, which is widely used against this disease in inaequalis is reported to encounter resistance development to India, other DMI fungicides like penconazol, flusilazole and DMI fungicides in other countries (Thindet al ., 1986), it is fenarimol are also applied. In the recent years, azoxystrobin necessary to initiate monitoring programmes for determining has also been introduced for the control of powdery mildew changes in the sensitivity levels ofV. inaequalis populations and also downy mildew in grapevine. Conidial populations of to these fungicides. A simple and quick method based on this fungus collected from various regions during 1995-97 spore germination, germ tube length and morphology has were studied for detection of resistant strains. The first case of been developed which can beeffectively used for determining development of resistance inU. necator to triadimefon was resistanceto DMI fungicides (Thindet al., 1987). reported byThindetal . (1998) in India. CONCLUSIONSAND FUTURE OUTLOOK Fifteen populations ofU. necator , each obtained from five infected leaves bearing profuse sporulation, were collected Several site specific fungicides are used in Indian agriculture from treated vineyards in Punjab, Maharashtra and for managing different crop diseases. Reports of resistance Karnataka. These were studied for determining their build up to some commonly used fungicides in field sensitivity levels to triadimefon (Bayleton 25) following populations of certain pathogens indicates the likely risk criteria of conidial germtube length and sporulation on leaf these new generation fungicides may pose in managing plant discs treated with different concentrations ranging from 0.01 diseases more effectively. Risk assessment is crucial for the to 10 g/ml of this fungicide. Criterion of germtube length of newly developed fungicides before these are introduced for more than 250 at 0.3 g/ml or above was taken for determining the commercial use by the farmers. New research initiatives resistance to triadimefon (Steva and Clerjeau, 1990 ; Thind need to be developed to predict the actual risk of resistance. T.S. Thind 61 New technologies are required for monitoring the Aspergillus flavusto certain fungicides. ISPP Chemical performance of resistance management strategies and to help Control Newsletter 6 : 23. predict problems before they occur. New techniques based on Gangawane, L.V., Kamble, S.S. and Arora, R.K. 1995. molecular biology may prove handy for rapid detection of Synergistic effect of other fungicides on metalaxyl resistance in pathogen population and may provide necessary resistant isolates ofPhytophthora infestans from Nilgiri information about performance of an anti-resistance strategy. Hills.Indian Journal of Plant Protection 23 : 159-162. 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