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.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

Show more authors

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

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 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

1327 Accesses

58 Citations

2 Altmetric

Metricsdetails

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.

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.

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,

Having regard to the Treaty on the Functioning of the European Union,

Having regard to Regulation (EC) No 396/2005 ( 1 ) of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC, and in particular Article 14(1)(a) and Article 49(2) thereof,

Whereas:

(1) For captan, carbendazim, cyromazine, ethephon, fena­miphos, thiophanate-methyl, triasulfuron and triti­conazole maximum residue levels (MRLs) are set in Annex II and Part B of Annex III to Regulation (EC) No 396/2005.

(2) For captan the Commission was informed that uses on celery, spinach, and parsley were revoked and thus the corresponding MRLs could be reduced without requiring the opinion of the European Food Safety Authority, here­inafter ‘the Authority’, in accordance with Article 17 of Regulation (EC) No 396/2005.

(3) For cyromazine an evaluation by the Authority ( 2 ) indicated that the MRL for lettuce may raise concerns of consumer protection. The Authority recommended lowering that MRL. These concerns also apply to scarole.

(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 that it is necessary to lower the MRLs as regards carbendazim for grapefruits, oranges, and tomatoes and as regards thiophanate-methyl for tomatoes.

(5) For ethephon ( 5 ), fenamiphos ( 6 ), triasulfuron ( 7 ) and triti­conazole ( 8 ), the Authority submitted reasoned opinions on the existing MRLs in accordance with Article 12(2) of Regulation (EC) No 396/2005. The Authority concluded that it is necessary to lower the MRLs as regards tria­sulfuron for barley, oats, rye, and wheat and as regards fenamiphos for tomatoes, aubergines, peppers, water 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 the MRLs on new commodities for these four substances, temporarily set in Part B of Annex III to Regulation (EC) No 396/2005, to Annex II to that Regulation.

(6) Based on the reasoned opinions of the Authority and taking into account the factors relevant to the matter under consideration, the appropriate modifications to the MRLs fulfil the requirements of Article 14(2) 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.

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

( 1 ) OJ L 70, 16.3.2005, p. 1. ( 2 ) EFSA scientific report (2008) 168.

( 3 ) EFSA Scientific Report (2009) 289. ( 4 ) See footnote 3. ( 5 ) EFSA Journal (2009) 7(10): 1347. ( 6 ) EFSA scientific report (2009) 331. ( 7 ) EFSA scientific report (2009) 278. ( 8 ) EFSA scientific report (2009) 277.

(8) A reasonable period should be allowed to elapse before the modified MRLs become applicable in order to permit Member States and interested parties to prepare them­selves to meet the new requirements which will result from the modification of the MRLs.

(9) Annex II to Regulation (EC) No 396/2005 and Part B of Annex III to that Regulation should therefore be amended accordingly.

(10) In order to allow for the normal marketing, processing and consumption of products, this Regulation should provide for a transitional arrangement for products which have been lawfully produced before the modifi­cation of the MRLs and for which information shows that a high level of consumer protection is maintained.

(11) The measures provided for in this Regulation are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health and neither the European Parliament nor the Council has opposed them,

HAS ADOPTED THIS REGULATION:

Article 1

Annexes II and III to Regulation (EC) No 396/2005 are amended as follows:

(1) Annex II is amended in accordance with the Annex to this Regulation.

(2) In Part B of Annex III, the columns for ethephon, fena­miphos, triasulfuron and triticonazole are deleted.

Article 2

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 products which were lawfully produced before 1 January 2012:

(a) captan: celery, spinach and parsley;

(b) carbendazim and thiophanate-methyl: frozen, canned, preserved and processed products of grapefruits, oranges and tomatoes;

(c) fenamiphos: fruiting vegetables, bananas, oilseeds and Brussels sprouts.

Article 3

This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the European Union.

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

EN L 152/2 Official Journal of the European Union 11.6.2011

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 triti­conazole are replaced by the following:

EN 11.6.2011 Official Journal of the European Union L 152/3

EN L 152/4

Official Journal of the European U

nion 11.6.2011

‘Pesticide residues and maximum residue levels (mg/kg)

Code number Groups and examples of individual products to which the MRLs apply (a )

Capt

an

Car

bend

azim

and

beno

myl

(sum

of

beno

myl

and

carb

enda

zim

expr

esse

das

ca

rben

dazi

m)

(R)

Cyro

maz

ine

Ethe

phon

Fena

mip

hos

(sum

offe

nam

ipho

san

dits

su

lpho

xide

and

sulp

hone

expr

esse

das

fe

nam

ipho

s)

Thio

phan

ate-

met

hyl(

R)

Tria

sulfu

ron

Triti

cona

zole

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0100000 1. FRUIT FRESH OR FROZEN; NUTS 0,05 (*) 0,05 (*) 0,01 (*)

0110000 (i) Citrus fruit 0,02 (*) 0,05 (*) 0,02 (*)

0110010 Grapefruit (Shaddocks, pomelos, sweeties, tangelo (except mineola), ugli and other hybrids)

0,2 6

0110020 Oranges (Bergamot, bitter orange, chinotto and other hybrids) 0,2 6

0110030 Lemons (Citron, lemon) 0,7 6

0110040 Limes 0,7 6

0110050 Mandarins (Clementine, tangerine, mineola and other hybrids) 0,7 6

0110990 Others 0,1 (*) 0,1 (*)

0120000 (ii) Tree nuts (shelled or unshelled) 0,1 (*) 0,02 (*) 0,2 (*)

0120010 Almonds 0,3 0,1

0120020 Brazil nuts 0,02 (*) 0,1

0120030 Cashew nuts 0,02 (*) 0,1

0120040 Chestnuts 0,02 (*) 0,1

0120050 Coconuts 0,02 (*) 0,1

0120060 Hazelnuts (Filbert) 0,02 (*) 0,2

EN 11.6.2011

Official Journal of the European U

nion L 152/5

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0120070 Macadamia 0,02 (*) 0,1

0120080 Pecans 0,02 (*) 0,1

0120090 Pine nuts 0,02 (*) 0,1

0120100 Pistachios 0,02 (*) 0,1

0120110 Walnuts 0,02 (*) 0,5

0120990 Others 0,02 (*) 0,1

0130000 (iii) Pome fruit 3 (+) 0,02 (*)

0130010 Apples (Crab apple) 0,2 0,6 0,5

0130020 Pears (Oriental pear) 0,2 0,05 (*) 0,5

0130030 Quinces 0,2 0,05 (*) 0,5

0130040 Medlar (**) (**) 0,05 (*) (**)

0130050 Loquat (**) (**) 0,05 (*) (**)

0130990 Others 0,2 0,05 (*) 0,5

0140000 (iv) Stone fruit 0,02 (*)

0140010 Apricots 3 0,2 0,05 (*) 2

0140020 Cherries (sweet cherries, sour cherries) 5 0,5 3 0,3

0140030 Peaches (Nectarines and similar hybrids) 0,02 (*) 0,2 0,05 (*) 2

0140040 Plums (Damson, greengage, mirabelle, sloe) 1 0,5 0,05 (*) 0,3

0140990 Others 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0150000 (v) Berries & small fruit

0151000 (a) Table and wine grapes 0,02 (*) 0,03

0151010 Table grapes 0,3 0,7 0,1 (*)

0151020 Wine grapes 0,5 2 3

0152000 (b) Strawberries 3 (+) 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

EN L 152/6

Official Journal of the European U

nion 11.6.2011

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0153000 (c) Cane fruit 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0153010 Blackberries 3 (+)

0153020 Dewberries (Loganberries, boysenberries, and cloudberries) 0,02 (*)

0153030 Raspberries (Wineberries, arctic bramble/raspberry, (Rubus arcticus), nectar raspberries (Rubus arcticus x idaeus))

3 (+)

0153990 Others 0,02 (*)

0154000 (d) Other small fruit & berries 0,1 (*) 0,02 (*) 0,1 (*)

0154010 Blueberries (Bilberries) 0,02 (*) 20

0154020 Cranberries (Cowberries (red bilberries)) 0,02 (*) 0,05 (*)

0154030 Currants (red, black and white) 3 (+) 0,05 (*)

0154040 Gooseberries (Including hybrids with other ribes species) 3 (+) 0,05 (*)

0154050 Rose hips 0,02 (*) (**) (**) 0,05 (*) (**)

0154060 Mulberries (arbutus berry) 0,02 (*) (**) (**) 0,05 (*) (**)

0154070 Azarole (mediteranean medlar) (Kiwiberry (Actinidia arguta)) 0,02 (*) (**) (**) 0,05 (*) (**)

0154080 Elderberries (Black chokeberry (appleberry), mountain ash, buckthorn (sea sallowthorn), hawthorn, service berries, and other treeberries)

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

0154990 Others 0,02 (*) 0,05 (*)

0160000 (vi) Miscellaneous fruit 0,02 (*)

0161000 (a) Edible peel 0,02 (*) 0,1 (*) 0,1 (*)

0161010 Dates 0,05 (*)

0161020 Figs 0,05 (*)

0161030 Table olives 5 (+)

0161040 Kumquats (Marumi kumquats, nagami kumquats, limequats (Citrus aurantifolia x Fortunella spp.))

0,05 (*)

EN 11.6.2011

Official Journal of the European U

nion L 152/7

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0161050 Carambola (Bilimbi) (**) (**) 0,05 (*) (**)

0161060 Persimmon (**) (**) 0,05 (*) (**)

0161070 Jambolan (java plum) (Java apple (water apple), pomerac, rose apple, Brazilean cherry Surinam cherry (grumichama Eugenia uniflora))

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

0161990 Others 0,05 (*)

0162000 (b) Inedible peel, small 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0162010 Kiwi

0162020 Lychee (Litchi) (Pulasan, rambutan (hairy litchi), mangosteen)

0162030 Passion fruit

0162040 Prickly pear (cactus fruit) (**) (**) (**)

0162050 Star apple (**) (**) (**)

0162060 American persimmon (Virginia kaki) (Black sapote, white sapote, green sapote, canistel (yellow sapote), and mammey sapote)

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

0162990 Others

0163000 (c) Inedible peel, large

0163010 Avocados 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0163020 Bananas (Dwarf banana, plantain, apple banana) 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0163030 Mangoes 2 0,5 0,05 (*) 1

0163040 Papaya 0,02 (*) 0,2 0,05 (*) 1

0163050 Pomegranate 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0163060 Cherimoya (Custard apple, sugar apple (sweetsop), llama and other medium sized Annonaceae)

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

0163070 Guava (Red pitaya or dragon fruit (Hylocereus undatus)) 0,02 (*) (**) (**) 0,05 (*) (**)

0163080 Pineapples 0,02 (*) 0,1 (*) 2 0,1 (*)

EN L 152/8

Official Journal of the European U

nion 11.6.2011

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0163090 Bread fruit (Jackfruit) 0,02 (*) (**) (**) 0,05 (*) (**)

0163100 Durian 0,02 (*) (**) (**) 0,05 (*) (**)

0163110 Soursop (guanabana) 0,02 (*) (**) (**) 0,05 (*) (**)

0163990 Others 0,02 (*) 0,1 (*) 0,05 (*) 0,1 (*)

0200000 2. VEGETABLES FRESH OR FROZEN 0,05 (*) 0,01 (*)

0210000 (i) Root and tuber vegetables 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0211000 (a) Potatoes 0,05 1

0212000 (b) Tropical root and tuber vegetables 0,02 (*) 0,05 (*)

0212010 Cassava (Dasheen, eddoe (Japanese taro), tannia)

0212020 Sweet potatoes

0212030 Yams (Potato bean (yam bean), Mexican yam bean)

0212040 Arrowroot (**) (**) (**)

0212990 Others

0213000 (c) Other root and tuber vegetables except sugar beet

0213010 Beetroot 0,02 (*) 0,05 (*)

0213020 Carrots 0,1 1

0213030 Celeriac 0,1 0,05 (*)

0213040 Horseradish (Angelica roots, lovage roots, gentiana roots,) 0,02 (*) 0,05 (*)

0213050 Jerusalem artichokes 0,02 (*) 0,05 (*)

0213060 Parsnips 0,02 (*) 0,05 (*)

0213070 Parsley root 0,02 (*) 0,05 (*)

0213080 Radishes (Black radish, Japanese radish, small radish and similar varieties, tiger nut (Cyperus esculentus))

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

EN 11.6.2011

Official Journal of the European U

nion L 152/9

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0213090 Salsify (Scorzonera, Spanish salsify (Spanish oysterplant)) 0,02 (*) 0,05 (*)

0213100 Swedes 0,02 (*) 0,05 (*)

0213110 Turnips 0,02 (*) 0,05 (*)

0213990 Others 0,02 (*) 0,05 (*)

0220000 (ii) Bulb vegetables 0,02 (*) 0,1 (*) 0,05 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0220010 Garlic

0220020 Onions (Silverskin onions)

0220030 Shallots

0220040 Spring onions (Welsh onion and similar varieties)

0220990 Others

0230000 (iii) Fruiting vegetables

0231000 (a) Solanacea 1

0231010 Tomatoes (Cherry tomatoes, tree tomato, Physalis, gojiberry, wolfberry (Lycium barbarum and L. chinense))

2 (+) 0,3 1 0,04 1

0231020 Peppers (Chilli peppers) 0,1 0,1 (*) 0,05 (*) 0,04 0,1 (*)

0231030 Aubergines (egg plants) (Pepino) 0,02 (*) 0,5 0,05 (*) 0,04 2

0231040 Okra, lady's fingers 0,02 (*) 2 0,05 (*) 0,02 (*) 1

0231990 Others 0,02 (*) 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0232000 (b) Cucurbits - edible peel 0,02 (*) 0,1 (*) 1 0,05 (*) 0,02 (*) 0,1 (*)

0232010 Cucumbers

0232020 Gherkins

0232030 Courgettes (Summer squash, marrow (patisson))

0232990 Others

0233000 (c) Cucurbits-inedible peel 0,1 (*) 0,05 (*) 0,02 (*)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0233010 Melons (Kiwano) 0,1 0,3 0,3

0233020 Pumpkins (Winter squash) 0,02 (*) 0,05 (*) 0,5

0233030 Watermelons 0,02 (*) 0,3 0,3

0233990 Others 0,02 (*) 0,05 (*) 0,3

0234000 (d) Sweet corn 0,02 (*) 0,1 (*) 0,05 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0239000 (e) Other fruiting vegetables 0,02 (*) 0,1 (*) 0,05 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0240000 (iv) Brassica vegetables 0,02 (*) 0,05 (*) 0,05 (*) 0,02 (*)

0241000 (a) Flowering brassica 0,1 (*) 0,1 (*)

0241010 Broccoli (Calabrese, Chinese broccoli, broccoli raab)

0241020 Cauliflower

0241990 Others

0242000 (b) Head brassica

0242010 Brussels sprouts 0,5 1

0242020 Head cabbage (Pointed head cabbage, red cabbage, savoy cabbage, white cabbage)

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

0242990 Others 0,1 (*) 0,1 (*)

0243000 (c) Leafy brassica 0,1 (*) 0,1 (*)

0243010 Chinese cabbage (Indian (Chinese) mustard, pak choi, Chinese flat cabbage (tai goo choi), choi sum, peking cabbage (pe-tsai))

0243020 Kale (Borecole (curly kale), collards, Portuguese Kale, Portuguese cabbage, cow cabbage)

0243990 Others

0244000 (d) Kohlrabi 0,1 (*) 0,1 (*)

0250000 (v) Leaf vegetables & fresh herbs 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0251000 (a) Lettuce and other salad plants including Brassicacea

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0251010 Lamb's lettuce (Italian cornsalad) 0,02 (*) 15

0251020 Lettuce (Head lettuce, lollo rosso (cutting lettuce), iceberg lettuce, romaine (cos) lettuce)

0,02 (*) 3

0251030 Scarole (broad-leaf endive) (Wild chicory, red-leaved chicory, radicchio, curld leave endive, sugar loaf)

2 0,05 (*)

0251040 Cress 0,02 (*) 15

0251050 Land cress 0,02 (*) (**) (**) (**)

0251060 Rocket, Rucola (Wild rocket) 0,02 (*) 15

0251070 Red mustard 0,02 (*) (**) (**) (**)

0251080 Leaves and sprouts of Brassica spp (Mizuna, leaves of peas and radish and other babyleaf brassica crops (crops harvested up to 8 true leaf stage))

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

0251990 Others 0,02 (*) 15

0252000 (b) Spinach & similar (leaves) 0,02 (*)

0252010 Spinach (New Zealand spinach, amaranthus spinach) 0,05 (*)

0252020 Purslane (Winter purslane (miner’s lettuce), garden purslane, common purslane, sorrel, glassworth, Agretti (Salsola soda))

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

0252030 Beet leaves (chard) (Leaves of beetroot) 20

0252990 Others 0,05 (*)

0253000 (c) Vine leaves (grape leaves) 0,02 (*) (**) (**) (**)

0254000 (d) Water cress 0,02 (*) 0,05 (*)

0255000 (e) Witloof 0,02 (*) 0,05 (*)

0256000 (f) Herbs 0,02 (*) 15

0256010 Chervil

0256020 Chives

0256030 Celery leaves (Fennel leaves, Coriander leaves, dill leaves, Caraway leaves, lovage, angelica, sweet cisely and other Apiacea leaves)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0256040 Parsley

0256050 Sage (Winter savory, summer savory,) (**) (**) (**)

0256060 Rosemary (**) (**) (**)

0256070 Thyme (Marjoram, oregano) (**) (**) (**)

0256080 Basil (Balm leaves, mint, peppermint) (**) (**) (**)

0256090 Bay leaves (laurel) (**) (**) (**)

0256100 Tarragon (Hyssop) (**) (**) (**)

0256990 Others (Edible flowers)

0260000 (vi) Legume vegetables (fresh) 0,05 (*) 0,02 (*) 0,1 (*)

0260010 Beans (with pods) (Green bean (french beans, snap beans), scarlet runner bean, slicing bean, yardlong beans)

2 (+) 0,2 5

0260020 Beans (without pods) (Broad beans, Flageolets, jack bean, lima bean, cowpea)

2 (+) 0,1 (*) 0,05 (*)

0260030 Peas (with pods) (Mangetout (sugar peas, snow peas)) 0,02 (*) 0,2 5

0260040 Peas (without pods) (Garden pea, green pea, chickpea) 0,02 (*) 0,1 (*) 0,05 (*)

0260050 Lentils 0,02 (*) 0,1 (*) 0,05 (*)

0260990 Others 0,02 (*) 0,1 (*) 0,05 (*)

0270000 (vii) Stem vegetables (fresh) 0,1 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0270010 Asparagus 0,02 (*) 0,05 (*)

0270020 Cardoons 0,02 (*) 0,05 (*)

0270030 Celery 0,02 (*) 2

0270040 Fennel 0,02 (*) 0,05 (*)

0270050 Globe artichokes 0,02 (*) 2

0270060 Leek 2 0,05 (*)

0270070 Rhubarb 0,02 (*) 0,05 (*)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0270080 Bamboo shoots 0,02 (*) (**) (**) (**)

0270090 Palm hearts 0,02 (*) (**) (**) (**)

0270990 Others 0,02 (*) 0,05 (*)

0280000 (viii) Fungi 0,02 (*) 0,05 (*) 0,02 (*) 0,1 (*)

0280010 Cultivated (Common mushroom, Oyster mushroom, Shi-take) 1 5

0280020 Wild (Chanterelle, Truffle, Morel, Cep) 0,1 (*) 0,05 (*)

0280990 Others 0,1 (*) 0,05 (*)

0290000 (ix) Sea weeds 0,02 (*) (**) (**) 0,05 (*) 0,02 (*) (**)

0300000 3. PULSES, DRY 0,02 (*) 0,1 (*) 0,05 (*) 0,05 (*) 0,02 (*) 0,1 (*) 0,05 (*) 0,01 (*)

0300010 Beans (Broad beans, navy beans, flageolets, jack beans, lima beans, field beans, cowpeas)

0300020 Lentils

0300030 Peas (Chickpeas, field peas, chickling vetch)

0300040 Lupins

0300990 Others

0400000 4. OILSEEDS AND OILFRUITS 0,02 (*) 0,05 (*) 0,05 (*)

0401000 (i) Oilseeds 0,02 (*) 0,02 (*)

0401010 Linseed 0,1 (*) 0,1 (*) 0,1 (*)

0401020 Peanuts 0,1 (*) 0,1 (*) 0,1 (*)

0401030 Poppy seed 0,1 (*) 0,1 (*) 0,1 (*)

0401040 Sesame seed 0,1 (*) 0,1 (*) 0,1 (*)

0401050 Sunflower seed 0,1 (*) 0,1 (*) 0,1 (*)

0401060 Rape seed (Bird rapeseed, turnip rape) 0,1 (*) 0,1 (*) 0,1 (*)

0401070 Soya bean 0,2 0,1 (*) 0,3

EN L 152/14

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nion 11.6.2011

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0401080 Mustard seed 0,1 (*) 0,1 (*) 0,1 (*)

0401090 Cotton seed 0,1 (*) 2 (+) 0,1 (*)

0401100 Pumpkin seeds (Other seeds of cucurbitacea) 0,1 (*) 0,1 (*) 0,1 (*)

0401110 Safflower (**) (**) 0,1 (*) (**)

0401120 Borage (**) (**) 0,1 (*) (**)

0401130 Gold of pleasure (**) (**) 0,1 (*) (**)

0401140 Hempseed 0,1 (*) 0,1 (*) 0,1 (*)

0401150 Castor bean (**) (**) 0,1 (*) (**)

0401990 Others 0,1 (*) 0,1 (*) 0,1 (*)

0402000 (ii) Oilfruits 0,1 (*) 0,05 (*) 0,1 (*)

0402010 Olives for oil production 10 0,01 (*)

0402020 Palm nuts (palmoil kernels) (**) (**) 0,05 (*) (**) 0,02 (*)

0402030 Palmfruit (**) (**) 0,05 (*) (**) 0,02 (*)

0402040 Kapok (**) (**) 0,05 (*) (**) 0,02 (*)

0402990 Others 0,05 (*) 0,02 (*)

0500000 5. CEREALS 0,02 (*) 0,05 (*) 0,02 (*) 0,05 (*) 0,01 (*)

0500010 Barley 2 1 0,3

0500020 Buckwheat (Amaranthus, quinoa) 0,01 (*) 0,05 (*) 0,01 (*)

0500030 Maize 0,01 (*) 0,05 (*) 0,01 (*)

0500040 Millet (Foxtail millet, teff) 0,01 (*) 0,05 (*) 0,01 (*)

0500050 Oats 2 0,05 (*) 0,3

0500060 Rice 0,01 (*) 0,05 (*) 0,01 (*)

0500070 Rye 0,1 1 0,05

EN 11.6.2011

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0500080 Sorghum 0,01 (*) 0,05 (*) 0,01 (*)

0500090 Wheat (Spelt, triticale) 0,1 1 0,05

0500990 Others 0,01 (*) 0,05 (*) 0,01 (*)

0600000 6. TEA, COFFEE, HERBAL INFUSIONS AND COCOA 0,05 (*) 0,1 (*) 0,05 (*) 0,1 (*) 0,05 (*) 0,1 (*) 0,1 (*) 0,02 (*)

0610000 (i) Tea (dried leaves and stalks, fermented or otherwise of Camellia sinensis)

0620000 (ii) Coffee beans (**) (**) (**)

0630000 (iii) Herbal infusions (dried) (**) (**) (**)

0631000 (a) Flowers (**) (**) (**)

0631010 Camomille flowers (**) (**) (**)

0631020 Hybiscus flowers (**) (**) (**)

0631030 Rose petals (**) (**) (**)

0631040 Jasmine flowers (Elderflowers (Sambucus nigra)) (**) (**) (**)

0631050 Lime (linden) (**) (**) (**)

0631990 Others (**) (**) (**)

0632000 (b) Leaves (**) (**) (**)

0632010 Strawberry leaves (**) (**) (**)

0632020 Rooibos leaves (Ginkgo leaves) (**) (**) (**)

0632030 Maté (**) (**) (**)

0632990 Others (**) (**) (**)

0633000 (c) Roots (**) (**) (**)

0633010 Valerian root (**) (**) (**)

0633020 Ginseng root (**) (**) (**)

0633990 Others (**) (**) (**)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0639000 (d) Other herbal infusions (**) (**) (**)

0640000 (iv) Cocoa (fermented beans) (**) (**) (**)

0650000 (v) Carob (st johns bread) (**) (**) (**)

0700000 7. HOPS (dried), including hop pellets and unconcentrated powder 0,05 (*) 0,1 (*) 0,05 (*) 0,1 (*) 0,05 (*) 0,1 (*) 0,1 (*) 0,02 (*)

0800000 8. SPICES 0,05 (*) (**) (**) 0,1 (*) 0,05 (*) (**) 0,1 (*) 0,02 (*)

0810000 (i) Seeds (**) (**) (**)

0810010 Anise (**) (**) (**)

0810020 Black caraway (**) (**) (**)

0810030 Celery seed (Lovage seed) (**) (**) (**)

0810040 Coriander seed (**) (**) (**)

0810050 Cumin seed (**) (**) (**)

0810060 Dill seed (**) (**) (**)

0810070 Fennel seed (**) (**) (**)

0810080 Fenugreek (**) (**) (**)

0810090 Nutmeg (**) (**) (**)

0810990 Others (**) (**) (**)

0820000 (ii) Fruits and berries (**) (**) (**)

0820010 Allspice (**) (**) (**)

0820020 Anise pepper (Japan pepper) (**) (**) (**)

0820030 Caraway (**) (**) (**)

0820040 Cardamom (**) (**) (**)

0820050 Juniper berries (**) (**) (**)

0820060 Pepper, black and white (Long pepper, pink pepper) (**) (**) (**)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0820070 Vanilla pods (**) (**) (**)

0820080 Tamarind (**) (**) (**)

0820990 Others (**) (**) (**)

0830000 (iii) Bark (**) (**) (**)

0830010 Cinnamon (Cassia) (**) (**) (**)

0830990 Others (**) (**) (**)

0840000 (iv) Roots or rhizome (**) (**) (**)

0840010 Liquorice (**) (**) (**)

0840020 Ginger (**) (**) (**)

0840030 Turmeric (Curcuma) (**) (**) (**)

0840040 Horseradish (**) (**) (**)

0840990 Others (**) (**) (**)

0850000 (v) Buds (**) (**) (**)

0850010 Cloves (**) (**) (**)

0850020 Capers (**) (**) (**)

0850990 Others (**) (**) (**)

0860000 (vi) Flower stigma (**) (**) (**)

0860010 Saffron (**) (**) (**)

0860990 Others (**) (**) (**)

0870000 (vii) Aril (**) (**) (**)

0870010 Mace (**) (**) (**)

0870990 Others (**) (**) (**)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

0900000 9. SUGAR PLANTS 0,02 (*) (**) (**) 0,05 (*) (**) 0,05 (*) 0,01 (*)

0900010 Sugar beet (root) (**) (**) 0,1 (**)

0900020 Sugar cane (**) (**) 0,02 (*) (**)

0900030 Chicory roots (**) (**) 0,02 (*) (**)

0900990 Others (**) (**) 0,02 (*) (**)

1000000 10. PRODUCTS OF ANIMAL ORIGIN-TERRESTRIAL ANIMALS 0,02 (*) 0,05 (*) 0,01 (*)

1010000 (i) Meat, preparations of meat, offals, blood, animal fats fresh chilled or frozen, salted, in brine, dried or smoked or processed as flours or meals other processed products such as sausages and food preparations based on these

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

1011000 (a) Swine 0,05 (*) 0,02 (*)

1011010 Meat

1011020 Fat free of lean meat

1011030 Liver

1011040 Kidney

1011050 Edible offal

1011990 Others

1012000 (b) Bovine 0,05 (*) 0,02 (*)

1012010 Meat

1012020 Fat

1012030 Liver

1012040 Kidney

1012050 Edible offal

1012990 Others

1013000 (c) Sheep 0,02 (*)

1013010 Meat

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

1013020 Fat

1013030 Liver

1013040 Kidney

1013050 Edible offal

1013990 Others

1014000 (d) Goat 0,05 (*) 0,02 (*)

1014010 Meat

1014020 Fat

1014030 Liver

1014040 Kidney

1014050 Edible offal

1014990 Others

1015000 (e) Horses, asses, mules or hinnies (**) (**) 0,01 (*) (**)

1015010 Meat (**) (**) (**)

1015020 Fat (**) (**) (**)

1015030 Liver (**) (**) (**)

1015040 Kidney (**) (**) (**)

1015050 Edible offal (**) (**) (**)

1015990 Others (**) (**) (**)

1016000 (f) Poultry -chicken, geese, duck, turkey and Guinea fowl-, ostrich, pigeon 0,05 (*) 0,02 (*)

1016010 Meat

1016020 Fat

1016030 Liver

1016040 Kidney

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

1016050 Edible offal

1016990 Others

1017000 (g) Other farm animals (Rabbit, Kangaroo) (**) (**) 0,01 (*) (**)

1017010 Meat (**) (**) (**)

1017020 Fat (**) (**) (**)

1017030 Liver (**) (**) (**)

1017040 Kidney (**) (**) (**)

1017050 Edible offal (**) (**) (**)

1017990 Others (**) (**) (**)

1020000 (ii) Milk and cream, not concentrated, nor containing added sugar or sweetening matter, butter and other fats derived from milk, cheese and curd

0,05 (*) 0,02 (*) 0,05 (*) 0,005 (*) 0,05 (*)

1020010 Cattle

1020020 Sheep

1020030 Goat

1020040 Horse

1020990 Others

1030000 (iii) Birds' eggs, fresh preserved or cooked Shelled eggs and egg yolks fresh, dried, cooked by steaming or boiling in water, moulded, frozen or otherwise preserved whether or not containing added sugar or sweetening matter

0,05 (*) 0,2 0,05 (*) 0,01 (*) 0,05 (*)

1030010 Chicken

1030020 Duck (**) (**) (**)

1030030 Goose (**) (**) (**)

1030040 Quail (**) (**) (**)

1030990 Others (**) (**) (**)

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(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

1040000 (iv) Honey (Royal jelly, pollen) (**) (**) 0,05 (*) 0,01 (*) (**)

1050000 (v) Amphibians and reptiles (Frog legs, crocodiles) (**) (**) 0,05 (*) 0,01 (*) (**)

1060000 (vi) Snails (**) (**) 0,05 (*) 0,01 (*) (**)

1070000 (vii) Other terrestrial animal products (**) (**) 0,01 (*) 0,01 (*) (**)

(a ) For the complete list of products of plant and animal origin to which MRLs apply, reference should be made to Annex I. (*) Indicates lower limit of analytical determination

(**) Pesticide-code combination for which the MRL as set in Annex III Part B applies. (R) = The residue definition differs for the following combinations pesticide-code number: Carbendazim - code 1000000: Carbendazim and thiophanate-methyl, expressed as carbendazim Thiofanate-methyl - code 1000000: Carbendazim and thiophanate-methyl, expressed as carbendazim

Captan

(+) 0130000 (iii) Pome fruit Sum of captan and folpet.

(+) 0152000 (b) Strawberries Sum of captan and folpet.

(+) 0153010 Blackberries Sum of captan and folpet.

(+) 0153030 Raspberries (Wineberries, arctic bramble/raspberry, (Rubus arcticus), nectar raspberries (Rubus arcticus x idaeus))

Sum of captan and folpet.

(+) 0154030 Currants (red, black and white) Sum of captan and folpet.

(+) 0154040 Gooseberries (Including hybrids with other ribes species) Sum of captan and folpet.

(+) 0231010 Tomatoes (Cherry tomatoes, tree tomato, Physalis, gojiberry, wolfberry (Lycium barbarum and L. chinense))

Sum of captan and folpet.

(+) 0260010 Beans (with pods) (Green bean (french beans, snap beans), scarlet runner bean, slicing bean, yardlong beans)

Sum of captan and folpet.

(+) 0260020 Beans (without pods) (Broad beans, Flageolets, jack bean, lima bean, cowpea) Sum of captan and folpet.

Ethephon

(+) 0161030 Table olives MRL will be valid until 1 July 2011 pending submission and evaluation of additional residue trials

(+) 0401090 Cotton seed MRL will be valid until 1 July 2011 pending submission and evaluation of an additional metabolism study’

~ 1074 ~

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

E-ISSN: 2278-4136

P-ISSN: 2349-8234

JPP 2018; 7(5): 1074-1077

Received: 13-07-2018

Accepted: 15-08-2018

RM Dolas

M. Sc. Department Of Plant

Pathology and Agricultural

Microbiology, Post Graduate

Institute, Mahatma Phule Krishi

Vidyapeeth, Rahuri,

Ahmednagar Maharashtra, India

SB Gawade

Associate Professor of of

Microbiology, MPKV, Rahuri,

Maharashtra, India

MC Kasture

Associate Professor of 3Associate

Professor of SSAC, DBSKKV,

Dapoli, Maharashtra, India

Correspondence

RM Dolas

M. Sc. Department Of Plant

Pathology and Agricultural

Microbiology, Post Graduate

Institute, Mahatma Phule Krishi

Vidyapeeth, Rahuri,

Ahmednagar Maharashtra, India

Efficacy of seed treatment of fungicides, bio agents

and botanicals on seed mycoflora, seed germination

and seedling vigour index of mung bean

RM Dolas, SB Gawade and MC Kasture

Abstract

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.

The reduction in seed mycoflora due to this fungicidal treatment was 83.59 per cent over control. Among

the bioagents, Trichoderma viride @ 0.6 % showed 20.0 per cent seed mycoflora as against 51.2 per cent

in control. The reduction in seed mycoflora due to this bioagent treatment was 60.93 per cent over

control. Among botanicals, Garlic extract @ 0.5 % showed 21.5 per cent seed mycoflora as against 51.2

per cent in control. The reduction in seed mycoflora due to this botanical treatment was 58.0 per cent

over control. Further, the seed treatment with carbendazim @ 0.2 % showed 87 per cent seed germination

as against 69 per cent in control. The increase in seed germination due to this fungicidal treatment was

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

average productivity of 500 kg per hectare. Green gram output accounts for about 10-12 % of

total pulse production in the country. Mung production in the country remained stable more

than a decade through the 2000s at around 10 to 15 lakh tons. But a sudden jump in output was

noted in 2010-11 to 1.75 million tonnes primarily on account of rise in production from

Madhya Pradesh, Rajasthan and Tamil Nadu. In 2014-15 the mung bean production in India

was 1.39 million tonnes in which, Maharashtra’s contribution was about 20 %, while

Rajasthan was highest having 26 % of the total production.

~ 1075 ~

Journal of Pharmacognosy and Phytochemistry Mung bean production in the country is largely concentrated

in five states viz., Rajasthan, Maharashtra, Andhra Pradesh,

Gujarat and Bihar. These five states together contribute for

about 70 % of total Mung production in the country. There is

a distinct change in production pattern of mung bean across

states. Traditionally Rajasthan, Maharashtra, Andhra Pradesh

are major mung bean producing states. But there is significant

rise in production from other states in recent years

particularly, from Tamil Nadu, Uttar Pradesh and Gujarat.

Nevertheless production remained volatile across the years

with respect to most of the states. As per the latest available

estimates, Rajasthan, Maharashtra occupies the first two

positions, contributing over 45 %. Andhra Pradesh contributes

about 10 % while together Gujarat and Bihar account for

about 13 % of total production in the country (Anonymus,

2015).

Seed borne diseases are regarded as major constraints in

mung bean production. Infected seeds serve as the source for

the spread of the pathogen in disease free area. Seed infection

affects the import and export adversely because the seed

affected with microbes is not acceptable in international

market.

Seeds are the carrier of fungal flora either externally or

internally. The variety and intensity of fungal flora changes

area-wise and depends upon climate under which seed

produced storage or in field, if, not controlled. Also they

reduce seed quality i.e. seedling vigour and germination

percentage. So it is necessary to control harmful fungi before

causing damage by using suitable available measures i.e.

chemicals or bio agents.

The seed borne pathogens associated with seeds externally or

internally may cause seed rot, seedling blight and resulting

into low germination. Some fungi are associated with testa

and cotyledon of seeds infected in form of mycelium,

pycnidium and conidia or spores, after germination the

infection translation to hypocotyls and stem bases as well as

dicotyledonary leaves of seedling. Some fungal seed borne

pathogens having ability to kill the seedling or plants and

substantially, reduce the productive capacity (Shamsur

Rahman et al., 1999) [15]. Seed mycoflora play an important

role in determining the quality and longevity of seed.

As many as 16 diseases have been reported on mung bean,

many of these diseases have been reported as seed borne.

Seedling blight, root rot, stem rot, leaf and pod rot caused by

Macrophomina, Curvularia, Alternaria are some major fungal

diseases of mung bean. Species of Alternaria, Cladosporium,

Fusarium and Rhizoctonia are known to cause seed rot and

pre and post-emergence losses in green gram (Khare and

Chaubey, 1978; Saxena, 1986 and Patil et al., 1990) [14, 11].

Several workers reported Alternaria spp., Aspergillus flavus,

Aspergillus Niger, Cladosporium spp., Colletotrichum spp.,

Curvularia lunata, Fusarium spp., Macrophomina

phaseolina, Phoma medicaginis and Rhizopus are seed borne

and seed transmissible (Raut and Ahire, 1988; Patil et al.,

1990) [11]. The above mentioned fungi are potentially harmful

for cultivation of mung bean. So it is better to use some

protective measures to control these pathogens.

Material and Method

Mung bean seeds

To study the mycoflora associated with seeds of mung bean

and to test the efficacy of bio agents, botanicals and

fungicides on seed mycoflora, seed germination and seedling

vigour index, the seeds of mung bean varietiy Vaibhav were

collected from Pulses Improvement Project, Mahatma Phule

Krishi Vidyapeeth, Rahuri, Dist. Ahmednagar and Oilseed

Research Station, Jalgaon.

Glass wares

The standard corning brand glasswares viz., petriplates,

conical flasks, slides and test tubes were used.

Equipments

The laboratory equipments viz., autoclave, laminar flow

cabinet, incubator, sterio-binocular microscope, research

binocular microscope and weighing balance were used.

Incubation room

The incubation room was used for keeping the blotter plates.

The temperature of incubation room was 20 ± 20C controlled

automatically with alternate cycle of 12 hrs. light and 12 hrs

darkness (Automatically controlled by electronic timer).

Miscellaneous material

Pointed needles, inoculating needle, forceps, blotting papers,

scissor, glass marking pencil, glass rods, cover slips, towel

papers, mercuric chloride, spirit lamp and sterilized water etc.

were used.

Seed treatment with bio agents, botanicals and fungicides The bio agents i.e. Pseudomonas fluorescens @ 0.6 per cent

and Trichoderma viride @ 0.6 per cent alone were used to

find out their effect on seed mycoflora, seed germination and

seedling vigour index. Talc based formulations of these bio

agents were used for the seed treatment in mung bean. The

weight of talc based formulations of bio agents were taken on

weighing balance as per the dose and mixed with seeds of

mung bean. The material was slightly moistened with

sterilized water, shakes slightly so as to cover the entire seed

surface by bio agent and then was used for blotter test and

seed germination.

In botanical extracts, the crude extracts prepared from ginger

rhizome and garlic cloves were used for treatment to the

mung bean seeds. The concentration of crude extract was

taken with the help of sterilized pipette and mixed with seeds

so as to smear total surface of the seeds. Then this seed was

used for blotter test and seed germination.

In fungicidal seed treatments, required quantity of the

fungicides, on the basis of their concentration was weighted

accurately. The fungicide was mixed in seed, moistened with

sterilized water, shake the petridish containing seeds and

fungicides gently so as to cover the seed surface by fungicide.

After drying, the seeds were used for blotter test and seed

germination.

~ 1076 ~

Journal of Pharmacognosy and Phytochemistry Table 1: Fungicides, bioagents used and their botanicals

S. No. Fungicides, Bioagents, Botanicals Chemical name Active Ingredient Conc. Manufacturer

1. Captan 50 % WP N-(trichloromethyl thio-4)

Tetrachlorohexane-1-2-dicarboximide 50 % WP 0.2 % Rallis India Ltd. Mumbai

2. Carbendazim 50 % WP 2-(Methoxy arbonyl amino) benzimidazole 50 % WP 0.2 % BASF India Ltd., Mumbai

3.

Mancozeb 75 % WP

Manganese ethylene bisdithiocarbamate zink

sulphide 75 % WP 0.2 %

Indofil Chemicals Ltd.,

Mumbai

4. Carboxin (37.5 %) + Thiram (37.5

%) (Vitavax power)

5,6- dihydro-2- Methyl-1,4- oxathiin-3-

carboxamide (56) 75 % WP 0.2 % Uniroyal Chemical Co.

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 (%)

The per cent reduction in seed germination and seedling

vigour index with the inoculation of pathogens over control

was calculated. The data was subjected to statistical analysis

(Panse and Sukhatme, 1985)

Result and Discussion

The seed treatment of mung bean seed with carbendazim @

0.2 % was found most effective among all the seed

treatments. It showed 87 per cent seed germination as against

69 per cent in control treatment. The increase in seed

germination due to this fungicidal treatment was 20.68 per

cent over control. Among bio agents, 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 per

cent over control. The seed treatment of mung bean seed with

carbendazim @ 0.2 % was found most effective among all the

seed treatments. It showed 1592.1 seedling vigour index as

against 1166.1 in control treatment. The increase in seedling

vigour index due to this fungicidal treatment was 26.75 per

cent over control. Among bio agents, 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

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 treatment was 10.12 per cent over control.

The above results on seed germination and seedling vigour

index are in confirmation with Kulshreshta (1988), Pradeep et

al. (2000) [12], Krishnamurthy et al. (2003) [8], Akhtar et al.

(2005) [1], Dhutraj and Gokhale (2007) [5], Kar and Sahu

(2008) [6], Suryawanshi et al. (2008) [16], Koche et al. (2009) [7], Chilkuri and Giri (2014) [4], Chilkuri and Giri (2014) [4]

and Gawade et al. (2016). Kulshreshta (1988) tested efficacy

of thiram, bavistin, difolaton, dithane M-45 and cereson and

reported bavistin as the most effective fungicide for better

emergence, less seedling mortality and better yield in mung

bean. Pradeep et al. (2000) [12] recorded that seed treatment

with Trichoderma viride and Pseudomonas fluorescens

reduced the colonies of Aspergillus niger, Aspergillus flavus

and Fusarium moniliforme with significantly increased seed

germination and seedling vigour index in soybean.

Krishnamurthy et al. (2003) [8] reported that captafol and

bavistin gives effective control on Macrophomina phaseolina

and Fusarium spp. upto 98 %. Also Trichoderma harzianum

can control these fungi upto 92 % and improve seed

germination and vigour. Akhtar et al. (2005) [1] observed that

in mung bean, seed treatment with Carbendazim + Carbofuran

was highly effective against nematode and fungal disease

complex followed by seed powder of Azadirachta indica.

Dhutraj and Gokhale (2007) [5] reported that thiram and

bavistin was effective to control seed mycoflora of mung bean

than dithane M-45 and captan. Kar and Sahu (2008) [6] noted

that Trichoderma harzianum and Trichoderma viride

effectively control Macrophomina phaseolina. Also these

biocontrol agent enhanced germination and seedling vigour in

mung bean. Suryawanshi et al. (2008) [16] tested seven

fungicides against Macrophomina phaseolina of mung bean

and found that

carbendazim is most effective in both field and lab condition.

Koche et al. (2009) [7] reported that germination percentage

and seedling vigour index of soybean was increased with seed

treatment of Thiram + Carbendazim @ 3gm/kg each and also

reduced seed mycoflora. Chilkuri and Giri (2014) [4] studied

the seed treatment with talc based formulations of

Trichoderma viride and Pseudomonas fluorescens. These bio

agents were tested for their efficacy against seed mycoflora

and seed germination in green gram. Among these bio agents

T. viride was found superior in controlling the seed mycoflora

and also maximum seed germination was observed in T.

viride. Chilkuri and Giri (2014) [4] reported that seed

treatment with thiram + carbendazim (2:1) @ 3 g/kg of seed

was increasing the seed germination, shoot length, root length

and seedling vigour index in green gram and black gram.

Gawade et al. (2016) studied the efficacy of bio agents,

botanicals on seed mycoflora and seed quality in mung bean

and found that Trichoderma viride @ 0.6 % + Pseudomonas

fluorescens @ 0.6 % was most effective among the bio agents

and Garlic extract @ 1 % among botanicals was found most

effective in controlling seed borne pathogens and increasing

seed germination, seedling vigour index and field emergence

~ 1077 ~

Journal of Pharmacognosy and Phytochemistry 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)

S. No. Treatments Seed

germination (%)

Increase in seed germination

over control (%)

Seedling vigour

Index (SVI)

Increase in SVI over

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 1. Akhtar H, Ahmad V, Shukla PK. Comparative efficacy of

pesticides, bio-control agents and botanicals against

Fusarium oxysporum disease complex on Vigna mungo.

Ann. Pl. Protec. Sci. 2005; 13(2):434-437.

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

and black gram seeds. Sustainable technology

development in crop production, 1999, 1-3.

16. Suryawanshi AP, Gore DD, Gawade DB, Pawar AK,

Wadje AG. Efficacy of fungicides against Macrophomina

blight of mung bean. J Pl. Dis. Sci. 2008; 3(1):40-42.

INTRODUCTION

USE OFFUNGICIDES IN INDIA

Table 1

ACQUIRED RESISTANCE IN LABORATORY

Fungicides serve as important tools for managing diseases inagricultural crops. Although some plant diseases may bemanaged through resistant varieties and alteration of culturalpractices, several diseases are only managed acceptably withthe application of a suitable fungicide. About 150 differentchemicals belonging to different classes are used asfungicides in various countries including India.

Resistance to fungicides has become a challenging problemin the management of crop diseases and has threatened theperformance of some highly potent commercial fungicides(Brent, 1995). Unlike insecticides, where resistance problemsare known to occur much earlier, practical problems offungicide resistance has emerged much later in 1970's andthereafter. Worldwide, resistance in pathogen populations tomore than 100 different active ingredients has been reported.Incidence of resistance to fungicides has remained restrictedmainly to systemic fungicides that operate against single bio-chemical targets also known as single site inhibitors (Dekker,1985; Brent, 1995). These site-specific systemic fungicideswere introduced in the mid 1960's onwards and includeseveral major groups of fungicides such as benzimidazoles,pyrimidines, phenylamides, sterol biosynthesis inhibitors,dicarboximides, phenylamides, etc.. During the past decade,more novel compounds with different modes of actionnotably phenylpyrroles, anilinopyrimidines, strobilurins,spiroxamines, phenylpyridylanines, quinolines, etc. havebeen developed having bioefficacy against diverse plantdiseases. Several of these modern selective fungicides havebecome vulnerable to the risk of resistance development intarget pathogens in different countries (Brent and Hollomon,2007)

As compared to developed countries, not much work has beendone on the problem of fungicide resistance in India. Thiscould be attributed to the lack of awareness among the Indianworkers about the importance of the problem and non-availability of trained scientific manpower in this field. Mostof the earlier studies done on fungicide resistance in Indiapertained to acquired resistance using mutagens or training(pressurization) methods under laboratory conditions,without looking into their possible implications in practical

disease control. However, during the last two decades cases ofresistance development in field situations have also beenreported from different parts of the country (Thind, 2002;2008)

The use of fungicides in India is quite low as compared todeveloped countries. Overall fungicide use is less thaninsecticides and herbicides. The consumption of fungicides in2009 was 8307 MT compared to 26756 MT of insecticidesand 6040 MT of herbicides with market share at 19%compared to 61% of insecticides and 17 % of herbicides. Thecurrent fungicide market in India is worth Rs. 4300 millions.

Apart from conventional compounds like sulphur,dithiocarbamates, copper-based, mercurials, phthalimides,etc., several of the site-specific fungicides of the groups likebenzimidazoles, oxathiins, thiophanates, organophosphorus,triazoles and related sterol inhibitors, phenylamides,strobilurins and other recently developed compounds arebeing used in India for controlling different diseases on anumber of crops. As many as 52 fungicides belonging todifferent groups were registered for use in India ( ). Inaddition, formulations of combination products containingsystemic and contact fungicides are also registered.

Crop wise consumption of fungicides in India is maximum onpome fruits (12.7%), closely followed by potatoes (12.2%),rice (12.0%), tea (9.4%), coffee (8.0%), chillies (7.6%),grapevines (6.9%), other fruits (5.9%), other vegetables(4.6%) and other crops which account for about 75% of thetotal fungicides used in India (Thind, 2002).

In India, several cases of adaptive resistance to manyfungicides including multisite-action compounds have beenreported under laboratory conditions by different workers,but their possible implications in disease control have notbeen indicated. Various methods such as adaptation toincreasing fungicide concentrations, exposure to UVradiations and chemical mutagens have been employed tostudy resistance development in diverse fungi todithiocarbamates, copper-based, oxathiins, benzimidazoles,

KAVAKA 47: 54 - 62 (2016)

(Submitted on 05-03-2016 ;Accepted on 25-05-2016)

Development of Fungicide Resistance in Plant Pathogens with Reference to Indian ScenarioT.S.ThindDepartment of Plant Pathology, PunjabAgricultural University, Ludiana-141004, IndiaCorresponding authors E.mail :

ABSTRACTFungicides 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 disease control in many instances.The problem is more common with site-specific fungicides and performance of many of the systemic fungicides developed in the past threedecades has been adversely affected. Some of the fungicide groups such as benzimidazoles, phenylamides, dicarboximides and the recentlyintroduced 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. Thiscalls for implementation of suitable resistance management strategies to get expected disease control levels and to prolong the active life ofpotential fungicides.Keywords : Fungicides, resistance, plant pathogens, competitive fitness, pathogenic potential, site-specificity

54

organophosphorus, phenylamides, alkyl phosphonates,morpholines and antifungal antibiotics in the laboratory(Thind, 1995).

The increase in use of fungicides, particularly of selectivefungicides, on important crops caught the attention of someworkers about their likely effects on pathogen populationsand in the past years, cases of fungicide resistancedevelopment have also been reported under field conditionsin India (Thind, 2002; 2008). Sensitivity studies throughregular monitoring of conidial/sporangial populations ofseveral pathogens have led to the detection of fungicideresistant strains with low to high resistance levels in someplant pathogens. Some reported field cases of fungicideresistance in India are mentioned in and aredescribed in the following pages.

Apple scab caused by(Cke.) Wint. has become endemic in all the

important apple growing belts covering an area of 60, 000 hain Kashmir alone. As most of the commercial apple cultivarsare susceptible to scab, orchardists mainly depend onfungicides such as mancozeb, zineb, ziram, carbendazim,benomyl, captan, triazoles, etc. for its control. At least sixapplications of fungicides are recommended against thisdisease in Kashmir and seven in Himachal Pradesh (Guptaand Gupta, 1996) at various phenological stages starting fromsilver tip/green tip stage till harvest. But the growers usuallygive 12 -15 applications of different fungicides to ensure gooddisease control. Based on the observations by some growerson decreased level of disease control after prolonged andexclusive usage of mancozeb and carbendazim in their

orchards in Kashmir valley, sensitivity studies of conidialpopulations of from 40 affected orchards werecarried out. Apparently one isolate was obtained from eachorchard. The results were reported to indicate mancozebresistant strains in 12 orchards and carbendazim resistantstrains in 3 orchards (Basu Chaudhary and Puttoo, 1984).

Although apparently unusual, mancozeb resistant isolatescould tolerate 2.5 times higher levels of the fungicidecompared to sensitive isolates and the per cent disease controlranged from 41 to 77 in these orchards. The isolates provedpathogenic on young apple foliage of cv. Red Deliciousduring the first and second sub-culturings only. The resistancewas, however, found to be unstable as these isolates lost thecharacter after three sub-culturings on mancozeb freemedium and became non-sporulating. Since mancozebresistance in these isolates was not found to be stable by theworkers themselves and in any case was at a relatively lowlevel, the reduction in disease control over some years couldpossibly be attributed to the poorly managed spray schedulesof mancozeb. This fungicide stands low risk of resistancedevelopment in the pathogens due to its multi-site mode ofaction, and cases of practical resistance to mancozeb have notbeen reported elsewhere, despite its widespread use againstmany pathogens.

The carbendazim resistant isolates could tolerate 3-14 timeshigher levels of the fungicide. These are relatively lowresistance factors compared with carbendazim resistancereported elsewhere. In contrast to the mancozeb resistantisolates, the carbendazim resistance was found to be stable.The isolates retained the spore producing character and werepathogenic on young apple foliage during all sub-culture

RESISTANCE DEVELOPMENT IN FIELD

Table 2

Benzimidazoles

Apple scab ( ) :Venturia inaequalis V.Inaequalis

V. inaequalisTable 1. Site-specific fungicides registered for use against

Table 2. Reported cases of fungicide resistance under fieldsituations in India

T.S. Thind 55

Fungicide group Name of the fungicide

Oxathiins Carboxin, OxycarboxinBenzimidazoles Benomyl, CarbendazimGuanidines DodineThiophanates Thiophanate methylPhosphorothiolates Edifenphos, IprobenfosDicarboximides IprodioneAcylalanines Metalaxyl, Metalaxyl-M

(Mefenoxam)Cyano-acetamide oximes CymoxanilCinnamic acid derivatives DimethomorphTrizoles Propiconazole, Penconazole,

Myclobutanil, Triadimefon,Bitertanol, Hexaconazole,Difenoconazole, Tebuconazole,Flusilazole

Morpholines TridemorphPyrimidines FenarimolMelanin biosynthesisInhibitors

Tricyclazole, Carpropamid

Dithiolanes IsoprothiolaneStobilurins Azoxystrobin, Kresoxim

methyl, TrifloxystrobinOxazolidinones FamoxadoneImidazoles FenamidoneValinamides IprovalicarbAnilides ThifluzamideAntifungal antibiotics Aureofungin, Kasugamycin,

ValidamycinSource : Central Insecticides Board (www.cibrc.nic.in)

Fungicide Pathogen (Host) Reference

Carbendazim Venturia inaequalis(Apple)

Basuchaudhary andPutto (1984)

Gloeosporiumampelophagum (Grapes)

Kumar and Thind(1992)

Aspergillus flavus(Groundnut)

Gangawane andReddy (1985)

Cercospora beticola(Sugarbeet)

Pal andMukhopadhyay(1983)

Edifenphos Dreschlera oryzae (Rice) Annamalai andLalithakumari (1990)

Pyricularia oryzae (Rice) Lalithakumari andKumari (1987)

Metalaxyl Plasmopara viticola(Grape)

Rao and Reddy (1988)

Phytophthora infestans(Potato)

Arora et al.(1992)

Thind et al.(1999)

Phytophthora parasitica(Citrus)

Thind et al.(2009)

Pseudoperonosporacubensis (Cucumber)

Thind et al.(2011)

Oxadixyl Phytophthora infestans(Potato)

Singh et al.(1993)

Triadimefon Uncinula necator(Grape)

Thind et al.(1998)

inoculations. Strategies for the management of fungicideresistance in involving need-based applicationof fungicides and the use of sanitary, physical and culturalpractices to control the pathogen multiplication have beensuggested by Putto and Basu Chaudhary (1986).

Anthracnose, caused by (de Bary) Sacc.(syn. ), poses a serious threat to grapecultivation in Punjab and other parts of India and requiresregular fungicide applications for its control. A number oftreatments of benzimidazole and related fungicides likecarbendazim, benomyl and thiophanate methyl, as well asconvent ional contact fungicides (copper-based,dithiocarbamates, phthalimides, etc.) are applied repeatedlyby the growers to protect the plants from this disease. Due toexcessive and irrational use of benzimidazoles, developmentof resistance, associated with inferior disease control, hasbeen observed in and thestrains with high level of resistance to carbendazim have beenisolated from vineyards in the Punjab state (Kumar andThind, 1992). Studies were conducted during 1990-97 todetermine the population structure of withregard to fungicide sensitivity and strategies for itsmanagement.

In the preliminaryscreening a total of 80 isolates of collectedfrom various regions in the Punjab state during 1990-97 werestudied for their sensitivity to carbendazim (Bavistin 50 WP)using malt agar plates amended with 1 and 5 g/ml ofcarbendazim. Majority of the isolates showed sensitive orweakly resistant response and were unable to grow beyond 1g/ml of carbendazim. However, 36 % of the isolates showedgrowth at 5 g/ml indicating resistant response to carbendazim(Thind and Mohan, 1998). Twenty three isolates foundresistant in the preliminary screening were further grown athigher concentration up to 100 g/ml of carbendazim. Of these,all the isolates showed normal growth up to 50 g/ml while 15were able to grow even at a higher dose of 100 g/ml ofcarbendazim thus exhibiting high resistance factors (

). These isolates were obtained from vineyards receivingregular treatments of Bavistin and were mostly from areasnear Ludhiana. Resistance to carbendazim was found to bepersistent in nature as the resistant isolates were able to growat 50 g/ml of carbendazim even after one year of sub-culturingon fungicide-free medium. The isolates of

were categorised into three morphological groups andmajority of the resistant isolates produced reddish brown topeach red colonies (Thind ., 1994).

Further studies conducted from 2000-2004 revealed thatcarbendazim resistant isolates of werepersistent in natural populations and could be detectedfrequently in the vineyards around Ludhiana in Punjab state(Mohan ., 2005).

Pathogenicbehaviour of two resistant and two sensitive isolates wasstudied on detached leaves of cv. Perlette treated withdifferent concentrations of Bavistin in the laboratory. Whilethe sensitive isolates did not produce any symptoms above250 g/ml, both the resistant isolates Ga 28 and Ga 53developed normal sporulating lesions at 500 g/ml and alsoproduced mild symptoms even at 1000 g/ml of Bavistin thusconfirming their resistant character.

Cross resistance toother fungicides viz. Topsin-M (thiophanate methyl) , Captaf(captan), Indofil M-45 (mancozeb), Bordeaux mixture(copper sulphate + calcium hydroxide), and Bayleton- 5(triadimefon) was studied by growth inhibition assay as wellas by detached leaf assay by taking one resistant and onesensitive isolate. Observations revealed that resistant isolateGa 53 possessed cross resistance to Topsin-M which has asimilar mode of action as Bavistin (Mohan and Thind, 1995).On the other hand both resistant and sensitive isolatesexhibited sensitive response to all other fungicides tested. Inanother study ( Thind ., 1997) on cross resistance, threetriazole fungicides viz. Score (difenconazol), Corail(tebuconazol) and Olymp (flusilazole) and one pyridylaninecompound Dirango (fluazinam) were found to possess highinhibitory action against carbendazim resistant as well assensitive isolates with MIC values of triazoles rangingbetween 1-5 g/ml for both the types. Fluazinam also exhibitedgood efficacy at 10 g/ml and above. By detached leaf assaydifenconazole proved most effective and no symptomsdeveloped at 25 g/ml. Fluazinam arrested diseasesdevelopment completely at 500 g/ml by both the isolates andholds promise alongwith difenconazole to check resistance.

Indofil M-45 andBordeaux mixture to which carbendazim resistant isolates didnot show any cross resistance were tested in a resistanceaffected vineyard near Ludhiana. Bavistin (0.1%) when usedalone did not provide desired control of grape anthracnose. Incontrast, when it was applied in alternation with Bordeauxmixture (2:2:250) or Indofil M-45 (0.3%) there wassignificant reduction in disease severity (Mohan and Thind,1995). Triazole fungicides such as difenconazole,tebuconazole, flusilazole and fluazinam, a pyridylaninecompound, which showed promising efficacy againstresistant and sensitive isolates of inlaboratory studies using detached leaf assays (Thind ,1997; Thind and Mohan, 1998) are now used in fieldconditions as anti -resistance measures.

Application of an effective fungicide immediately after firstrain shower in March/April helps in checking the primary

V. inaequalis

G. ampelophagumElsinoe ampelina

Gloeosporium ampelophagum

G. ampelophagum

G. ampelophagum

G. ampelophagum

et al

G. ampelophagum

et al

et al

G. ampelophagumet al.

Grape anthracnose ( ):

:

Table3

:

:

:

Gloeosporium ampelophagum

Screening for carbendazim resistance

Pathogenic behaviour of resistant isolates

Cross resistance to other fungicides

Management of carbendazim resistance

Table 3. Structuring of isolatesfrom different vineyards for carbendazim sensitivity inPunjab (1990-1997)

Gloeosporium ampelophagum

Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario56

Number ofIsolatestested

ED50(?g /m l)

MIC(?g/ml)

Resistancefactor

Sensitivityclass

36 0.02-0.04 0.05-0.1 0.0 S21 0.04-0.16 0.20-0.5 1.8-4.5 WR8 14-50 40-100 360-900 HR15 69-100 > 100 > 900 HR

S = Sensitive, WR = Weekly resistant, HR = Highly resistantSource : Thind and Mohan (1998)

infection and multiplication of inoculum for subsequentinfections, thereby, reducing fungicide sprays andminimising the risk of resistance development to site-specificfungicides like carbendazim.

Leaf spot ofsugarbeet, caused by Sacc. is a serious diseaseproblem of sugarbeet in India. Various fungicides includingcarbendazim formulations are widely used to control thisdisease. Some natural populations of the fungus werescreened for sensitivity to carbendazim (Bavistin 50 WP). Itwas surprising to note that a natural mutant from untreatedfield was able to tolerate high concentrations of carbendazim(Pal and Mukhopadhyay, 1983).

Organophosphorusfungicides such as edifenphos and iprobenphos are widelyused in southern parts of India for the control of brown leafspot of rice caused by (Breda de Haan) Subram. andJain, which causes severe crop losses if not controlled in earlystages. Edifenphos (Hinosan) is used quite regularly in TamilNadu and other rice growing states for reducing crop lossesdue to this disease. Risk of resistance development toedifenphos has been determined in under selectionpressure of the fungicide in field during 1984 -1988 at avillage farm near Chingleput, Madras (Annamalai andLalithakumari, 1990). Field isolates of the pathogen werecollected to study the base-line data on the sensitivity beforecommencing the application of edifenphos in 1984 andsubsequently after the application of edifenphos every yearup to 1988. Repeated applications of edifenphos resulted inpatches of paddy crop cv. IR-50 with severe diseasemanifestation. The disease intensity in the treated plots wassurprisingly much higher than in untreated plots. Every year400 leaf samples were collected at random, the pathogen wasisolated (one lesion/plate) and screened for edifenphossensitivity by measuring the radial growth on PDA amendedwith 10, 20, 50, 200 and 300 g/ml of the fungicide. Tocharacterise the isolates for resistance, these were groupedinto four categories based on ED values i.e., sensitive, lowlevel resistance, moderate resistance and high level resistancehaving ED below 50, between 50-100, between 101-150 andabove 150 g/ml, respectively.

A shift in the level of sensitivity to edifenphos was noticedfrom year to year. In 1984, before the application ofedifenphos, 96 per cent of the isolates were sensitive toedifenphos at 50 g/ml, while in 1985, 1986, 1987 and 1988(i.e. after fungicide application), 74, 64, 50 and 48 per centrespectively of isolates showed the same level of sensitivity.The sensitivity data of the field isolates thus showed a clearshift in the level of sensitivity of due to frequentapplications of edifenphos ( ). Rate of uptake ofedifenphos was less in resistant strains of and thereduced membrane permeability was suggested as themechanism of resistance.

When tested for cross resistance to other fungicides,mancozeb showed significant inhibitory effect on the growthof edifenphos resistant isolates compared with the sensitive

strain and exhibited ED value of 45-50 M. Cross resistancewas observed to iprobenphos, an organophosphorus fungicidewith same mode of action. Other fungicides tested such ascopper oxychloride, benomyl, bitertanol, carbendazim andpyroquilon were less effective against resistant isolates andinhibited the growth of the fungus at higher concentrations(Annamalai and Lalithakumari, 1992). Cross resistancestudies and field treatments indicated that edifenphosresistance in can be counteracted by sprayingmancozeb as an alternative (replacement) fungicide.However, edifenphos is still used against and isworking well in most of the areas.

Blast disease of ricecaused by Cav. (syn. Cav. and

) is a serious disease in rice - growingareas of South Indian states. Edifenphos (Hinosan) is widelyused in foliar applications for the effective control of thisdisease. A preliminary study has been done to estimate thesensitivity of natural populations of isolates toedifenphos (Lalithakumari and Kumari, 1987). Diseasedleaves of rice cv. IR 50 were collected from fields sprayedregularly with edifenphos and fifty monosporic isolates ofthe pathogen were obtained. Sensitivity of these isolates toedifenphos was tested at ten concentrations ranging from 10to 100 m on oat meal agar by mycelial growth inhibitiontechnique. The ED values were compared with a sensitiveisolate, unexposed to edifenphos.

The sensitive isolate had ED value of 36.3 M. Seventeenisolates from treated fields showed a shift in their ED valuesabove 50 M and out of these 17 isolates, 9 isolates had EDvalues above 60 M (up to 75.8 M) thus indicating resistantresponse to edifenphos. Not much variation was observed inthe growth pattern, conidial morphology and pathogenicityamong the fifty isolates tested. The resistant isolates wereequally pathogenic when inoculated on one month oldseedlings of IR 50 rice cultivar. In cross resistance studies toother fungicides the resistant isolates showed positive crossresistance to iprobenphos, a related fungicide. Zirameffectively inhibited the growth of all the resistant isolates andits use was suggested as a companion fungicide in mixturewith edifenphos or as alternate spray fungicide(Lalithakumari and Mathivanan, 1990).

Grape downymildew caused by (Burk. & Curt.) Verl. & de Tonicauses severe losses in southern states of India such as

Sugarbeet leaf spot ( ) :

Organophosphorus Compounds

Brown spot of rice ( ) :

Table 4

Blast of rice

PhenylamidesGrape downy mildew ( ) :

Cercospora beticola

Drechslera oryzae

(Pyricularia oryzae) :

Plasmopara viticola

C. beticola

D. oryzae

D. oryzae

D. oryzae

D. oryzae

D. oryzae

D. oryzae

Pyricularia oryzae P. griseaMagnaporthe oryzae

P. oryzae

P. viticola

50

50

50

50

50

50

50

Table 4. Sensitivity range of field isolatesagainst edifenphos

Drechslera oryzae

T.S. Thind 57

ED50 Range(mg/ml)

Per cent of isolates1984 1985 1986 1987 1988

20 - 50 96 74 64 50 4851 - 100 4 26 16 18 16

101 - 150 0 0 20 18 10151 - 180 0 0 0 14 26

Source : Annamalai and Lalithakumari (1990)

Maharashtra, Andhra pradesh and Karnataka, especially ontwo commercial grape varieties Anab-e-Shahi and ThompsonSeedless affecting the production and quality of grapes. Whenmetalaxyl became available in late 1970s, it caught theattention of Indian farmers who found it miraculous incontrolling grape downy mildew which was earlier difficult tobe controlled by the traditional contact fungicides.

The grape growers around Hyderabad started using metalaxyl(Ridomil 25WP) in 1981 to control downy mildew. Based onreports by some grape growers in 1986 regarding the loss ofeffectiveness of metalaxyl in controlling grapevine downymildew in areas around Hyderabad, monitoring andsensitivity studies were undertaken for ascertaining the causefor reduced efficacy of metalaxyl (Rao and Reddy, 1988).Infected leaves were collected from three affected orchardssituated at three villages near Hyderabad which had receivedmetalaxyl applications for 3, 4 and 3 years, respectively.Sporangial populations from these samples were assayed forsensitivity to metalaxyl (Ridomil 25 WP) at 25, 50, 100 and250 g a.i./ml following detached leaf method of Pappas(1980). The preliminary sensitivity assays withpopulations indicated that the loss of efficacy of metalaxyl inthese vineyards was attributed to the development ofresistance to metalaxyl which had been used frequently by thegrowers (Rao and Reddy, 1988). Considerable difference inminimal inhibitory concentration was seen among the threepopulations.

Metalaxyl was almost completely inactive against pathogenpopulations collected from two of the three villages whichconfirmed the reports of grape growers regarding the loss ofefficacy of metalaxyl in controlling grape downy mildew( ). The continuous and exclusive use of metalaxyl(Ridomil 25 WP) by grape growers at these villages for 3-4years had led to the development of resistant populations of

. No further work has been done on this problem afterthis report. Several rounds of metalaxyl based combinationproducts with mancozeb viz. Ridomil-MZ (now RidomilGold) are used for the control of grape downy mildew inIndia. Although these combination products are known tominimise the risk of resistance development, regularmonitoring for determining the changes in sensitivity levelsof pathogen populations is necessary where these fungicidesare used frequently. A simple laboratory technique based onsporulation on leaf discs has been developed for laboratorytesting of fungicides (Thind ., 1988) which requires lessspace and can be easily employed for determining fungicideresistance in a large number of populations.

For themanagement of late blight of potato caused by(Mont.) de Bary, traditional fungicides such as mancozeb,zineb, copper oxychloride, chlorothalonil, etc. have been inuse since many years in India. However, these fungicidesprovided poor disease control under heavy disease pressure.The introduction of phenylamide fungicides provided muchneeded relief to the Indian farmers as these provided excellentcontrol of late blight even under severe disease conditions.Metalaxyl in combination with mancozeb (Ridomil MZ72WP) was commercially introduced in India during autumn1988 and since then is being widely used for the control of lateblight in different potato growing areas of the country. NowRidomil Gold having metalaxyl-M (also called mefenoxam)has been introduced recently in India for control of late blight.Although the mixture fungicides are expected to delay theonset of resistance build up, their use does not guaranteeprevention of resistance development (Gisi and Staehle-Csech, 1989).

Following the reports of resistance development to metalaxylin other countries ( Davidse ., 1981; Davidse, 1987),monitoring for metalaxyl resistant strains of wascarried out in Nilgiri hills from 1989-91 (Arora 1992).Sporangial populations of collected from potatofields sprayed with Ridomil MZ were analysed for theirresponse to metalaxyl by detached leaf method. Metalaxylresistant isolates of the pathogen were absent from early tomid summer potato crop seasons. These, however appearedtowards the end of summer season starting from last week ofJuly and a maximum frequency of 13% in the autumn.Variations in tolerance to metalaxyl from 50 to 700 g/ml wereobserved among different isolates resistant to metalaxyl.

The highly tolerant isolates (300 to 900 g/ml) were observedonly during the autumn season and comprised up to 6% of thetotal samples examined. The resistant isolates could beobtained in plots with a combined spray of metalaxyl andmancozeb and also in plots with individual sprays ofmancozeb or chlorothalonil and the control plots later duringthe season. The resistant isolates were found to be moreaggressive in traits like short incubation period, quickgermination of sporangia to zoospores, and ability to causelarger lesions, as compared to the sensitive isolates (Arora,1994). In another study of resistant monitoring in Nilgiri Hillsfollowing leaf disc assay, Gangawane (1995) havereported that out of isolates of tested, 82% weresensitive, 5% were moderately resistant ( RF 15-40) and 4%were highly resistant ( RF 60-70). Use of metalaxyl in mixturewith chlorothalonil was highly effective against bothsensitive and metalaxyl resistant isolates. Resistance tometalaxyl has also been reported in from Shimlahills after 5 years of use of Ridomil MZ by Singh . (1993)but the resistance level reported is quite low. They have alsoreported isolates of this pathogen developing moderateresistance to oxadixyl under experimental conditions.

In the Punjab state of India, metalaxyl in mixture withmancozeb (Ridomil MZ, Matco 8-64) is being widely usedfor the control of late blight of potato since 1989. Quite often,farmers also use self-prepared mixtures (tank-mixed) of

P. viticola

P.viticola

et al

P. viticola

P. infestans

et alP. infestans

et al.,P. infestans

et al.P. infestans

P. infestanset al

Table 5

Potato late blight ( ) :Phytophthora infestans

Table 5. Sensitivity of populations tometalaxyl by detached leaf assay

Plasmopara viticola

Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario58

ED50 Range(mg/ml)

Per cent of isolates1984 1985 1986 1987 1988

20 - 50 96 74 64 50 4851 - 100 4 26 16 18 16

101 - 150 0 0 20 18 10151 - 180 0 0 0 14 26

Source : Annamalai and Lalithakumari (1990)

metalaxyl (35% SD) and mancozeb in various proportions.Sensitivity levels of 68 populations collectedduring 1996-1999 crop seasons from various fields treatedwith fungicides in Punjab were monitored for their sensitivityto metalaxyl following detached leaf method (Thind

1989). Thirty one populations, mostly from Hoshiarpurdistrict, showed mild to severe infection at 10 g/ml, while 12populations, collected mostly during 1998-99 showedvarying levels of infection at 50 g/ml in the initial screening.When tested at higher concentraions of metalaxyl, threepopulations were able to produce symptoms at 100 and 200g/ml thus showing higher resistant response to metalaxyl(Thind 2001). The resistance factors of populationswith varying levels of decreased sensitivity to metalaxylranged between 2.8 to 28.5.Amarked decrease in the efficacyof Ridomil MZ was also observed in the field from wherehighly resistant populations were collected.

During 2005-2008 crop seasons, 48 sporangial populationsof collected from different potato growing areasin Punjab were tested for metalaxyl sensitivity among these10 populations showed resistant response causing infection at200 µg/ml of metalaxyl with resistance factor up to 60 (Kaur

2010). The resistant population exihibited competitivefitness in a mixture with sensitive population ( ).RAPD analysis of metalaxyl resistant populations of

was done with 10 oligonucleotide primers. Of 50primers initially used for amplification, 23 showed

polymorphism and 10 were able to distinguish resistant andsusceptible populations producing 2-3 unique bands.Information on banding pattern for all the primers was used todetermine genetic distance between resistant and sensitiveisolates and to construct a dendrogram. RAPD datadistinguished the test isolates into two groups thus separatingthe resistant and susceptible isolates. Using primer P 9 aunique band of 100bp was found in susceptible (S) isolatesindicating that these isolates are different from resistant onesin this 100bp region ( ).

The resistant populations were found to be highly pathogenicwhen inoculated on potato leaves of cv. Kufri Chandramukhi.Their disease severities (78 - 90 per cent) were comparablewith those of sensitive populations (82 - 100 per cent).Incubation period varied from 4 to 5 days in both resistant andsensitive populations. Sporulation was also comparable inboth types of populations. No cross resistance was observedto dimethomorp, mandipropamid, cymoxanil, benalaxyl,previcur, fluopicolide, azoxystrobin and multisite contactfungicides chlorothalonil, fluazinam and mancozeb.Dimethomorph has been reported to be effective incontrolling late blight of potato and tomato caused either by

metalaxyl sensitive or tolerant strains of (Cohen1995). Metalaxyl resistance was effectively managed

under field conditions through application of novel actionfungicides such as Infinito 68.75 SC (fluopicolide+propamocarrb chloride), Amistar 25 SC (azoxystrobin),Acrobat 50 WP (dimethomorph), Mandipropamid 250 SCand Curzate M-8 72 WP (cymoxanil + mancozeb).Combination of fungicides with different modes of actionretards development of resistance and ensure sustainablemanagement of late blight. The potential of several of thesenew fungicides has been documented in a recent review(Stevenson, 2009).

Phenylamide fungicides are regularly used by farmers invarious states of India including Punjab to manage downymildew infection on cucurbits such as cucumber and melons.Sensitivity changes to metalaxyl in

populations collected from cucumber andmuskmelon fields were monitored during 2007 and 2008.Maximum number of sporangial populations (12 out of 25)exhibited resistant response were collected in districtAmritsar with ED values of metalaxyl ranging between 30-150 g/ml and resistance factor (RF) between 6-30. Most of thepopulations from Jalandhar and Kapurthala showed normalsensitive response. Resistant populations possessed normalpathogenic potential and exhibited strong competitive fitnesswhen inoculated in mixture with sensitive populations (). Resistant populations did not show cross resistance to

fungicides with different modes of action such asazoxystrobin, cymoxanil, dimethomorph, fluopicolide,propamocarb, chlorothalonil and mancozeb. Thesefungicides were also found effective against metalaxylresistant populations under field condition and can form a partof the strategy to manage metalaxyl resistance in practice(Thind .,2011)

: Metalaxyl basedfungicides are commonly used to manage foot rot of citrus (

) in different states of India. A significant reductionin fungicide efficacy has been observed in many orchardsover the years. In Punjab state, investigations were carried out

P. infestans

etal.,

et al.,

P. infestans

et al.,

P.infestans

P. infestanset al.,

Pseudoperonosporacubensis

et al

P.parasitica

Table 6

Fig.1

Cucumber downy mildew ( ) :

Table7

Citrus foot rot ( )

Pseudoperonospora cubensis

Phytophthora parasitica

90

Fig. 1 Metalaxyl resistant and sensitive strains ofshowing amplification with P9Phytophthora infestans

Source: Kaur .(2010)et al

M R S

Table 6. Competitive fitness of metalaxyl resistant andsensitive populations of Phytophthora infestans

T.S. Thind 59

Metalaxylconcs. (mg/ml)

PDI with different combinations of R and S populationsR S R (50): S (50) R (25): S (75) R (75): S (25)

0 86.0 85.0 74.8 74.1 75.610 65.3 13.4 38.5 29.3 71.150 49.2 0.0 31.2 23.3 46.6100 44.2 0.0 22.1 7.2 33.5

R = Resistant strain, PI-24; S = Sensitive strain, PI-31; PDI=Per cent disease indexSource : Kaur et al. (2010)

to determine changes in sensitivity levels ofisolates from different citrus orchards where reduced efficacyhave been reported after metalaxyl applications. Of the 56isolates of the fungus tested, 9 isolates showed resistantresponse with ED values of metalaxyl ranging between 38-200 g/ml. Pathogenic potential, colony growth andsporulation of the resistant isolates were comparable withsensitive isolates (Thind ., 2009). The resistant isolatesdid not show cross resistance to azoxystrobin, cymoxanil,fluopicolide and previcur. Cymoxanil is providing effectivecontrol of foot rot in the orchards where metalaxyl resistancehas been a problem. A leaf disc assay involving fungicidetreated leaf discs of rough lemon rootstock placed on soilslurry has been developed for early detection of metalaxylresistance in citrus orchards (Thind , 2015).

Powderymildew, incited by (Schw.) Burr., is anotherserious disease of grapevine in India causing more damage tothe developing berries. For the past 20 years, various DMIfungicides, in addition to the traditional sulphur and dinocap,are being used for controlling this disease. Apart fromtriadimefon, which is widely used against this disease inIndia, other DMI fungicides like penconazol, flusilazole andfenarimol are also applied. In the recent years, azoxystrobinhas also been introduced for the control of powdery mildewand also downy mildew in grapevine. Conidial populations ofthis fungus collected from various regions during 1995-97were studied for detection of resistant strains. The first case ofdevelopment of resistance in to triadimefon wasreported byThind . (1998) in India.

Fifteen populations of , each obtained from fiveinfected leaves bearing profuse sporulation, were collectedfrom treated vineyards in Punjab, Maharashtra andKarnataka. These were studied for determining theirsensitivity levels to triadimefon (Bayleton 25) followingcriteria of conidial germtube length and sporulation on leafdiscs treated with different concentrations ranging from 0.01to 10 g/ml of this fungicide. Criterion of germtube length ofmore than 250 at 0.3 g/ml or above was taken for determiningresistance to triadimefon (Steva and Clerjeau, 1990 ; Thind

and Mohan, 1995). Majority of the populations showedtypical sensitive reaction as their germtubes measured lessthan 250 at 0.3 g/ml which was taken as the discriminatoryconcentration to distinguish resistant strains in the conidialpopulations. Germtubes of such conidia were distorted anddeformed at the tips and comparable in their response with thereference sensitive strainAne-17. However, three populationsshowed 4 to 6 per cent conidia with normal germtubesmeasuring more than 250 at a higher concentration of 3 g/mlof triadimefon. Two of these populations, 1a from Bangaloreand 7a from Pune, showed one per cent conidia producingnormal germtubes at a still higher concentration of 10 g/mlthus demonstrating low to moderate levels of resistancedevelopment in these populations (Thind ., 1998). Thesethree populations also developed sporulating colonies on theleaf discs at 3 and 10 g/ml thus confirming resistance totriadimefon. However, no apparent decline in the fieldperformance of this fungicide was observed except in thevineyard at Bangalore where a reduced disease control wasrecorded.

One isolate eachfrom populations 1a and 7a, showing moderate resistance totriadimefon, was further studied for sensitivity to two othersterol inhibiting fungicides, triadimenol (Baytan 5) andfenarimol (Rubigan 4) by sporulation test on treated leafdiscs. Compared to the sensitive strainAne-17, which showednegligible sporulation at 0.3 g/ml of triadimenol, isolate 1aand 7a produced some sporulation even at 1 g/ml thusconfirming cross resistance to triadimefon. However, onfenarimol treated discs, the two isolates were found to beequally sensitive as the reference strain, thereby, showing nocross resistance to fenarimol (Thind ., 1998). Fenarimolis now being used where reduced sensitivity has beenobserved to triazole fungicides.

Triazoles and other sterol inhibiting fungicides such asbitertanol (Baycor), hexaconazole (Contaf), myclobutanil(Systhane), penconazole (Topas) and fenarimol (Rubigan) arealso being used at present for controlling apple scab in India.Reduced sensitivity to these fungicides in this pathogen hasnot yet been reported from Indian orchards Since

is reported to encounter resistance development toDMI fungicides in other countries (Thind ., 1986), it isnecessary to initiate monitoring programmes for determiningchanges in the sensitivity levels of populationsto these fungicides. A simple and quick method based onspore germination, germ tube length and morphology hasbeen developed which can be effectively used for determiningresistance to DMI fungicides (Thind 1987).

Several site specific fungicides are used in Indian agriculturefor managing different crop diseases. Reports of resistancebuild up to some commonly used fungicides in fieldpopulations of certain pathogens indicates the likely riskthese new generation fungicides may pose in managing plantdiseases more effectively. Risk assessment is crucial for thenewly developed fungicides before these are introduced forthe commercial use by the farmers. New research initiativesneed to be developed to predict the actual risk of resistance.

P. parasitica

et al

et al.

U. necator

U. necatoret al

U. necator

et al

et al

Venturiainaequalis

et al

V. inaequalis

et al.,

50

TriazolesGrape powdery mildew ( ) :

:

CONCLUSIONSAND FUTURE OUTLOOK

Uncinula necator

Cross resistance to other SBI fungicides

Table 7. Pathogenic potential of metalaxyl resistant (R) andsensitive (S) population of Pseudoperonosporacubensis

Development of Fungicide Resistance in Plant Pathogens with Reference to Indian Scenario60

P.cubensispopulation

MIC(mg/ml)

Incubationperiod(Days)

Diseaseseverity

(%)

Sporulation(spores/cm2)

DM-12 (R) 100 5 80 75.5 × 103

DM-13 (R) 150 4 85 73.0 × 103

DM-15 (R) 10 4 90 75.0 × 103

DM-16 (R) 10 4 80 72.0 × 103

P.cubensispopulation

MIC(mg/ml)

Incubationperiod(Days)

Diseaseseverity

(%)

Sporulation(spores/cm2)

DM-12 (R) 100 5 80 75.5 × 103

DM-13 (R) 150 4 85 73.0 × 103

DM-15 (R) 10 4 90 75.0 × 103

DM-16 (R) 10 4 80 72.0 × 103

R=Resistant population, S= Sensitive populationSource : Thind et al. (2011)

New technologies are required for monitoring theperformance of resistance management strategies and to helppredict problems before they occur. New techniques based onmolecular biology may prove handy for rapid detection ofresistance in pathogen population and may provide necessaryinformation about performance of an anti-resistance strategy.Apart from using at-risk fungicides in mixture or alternationwith compounds from different modes of action, theirintegration with other control methods may help greatly inresistance management by keeping disease pressure at lowlevel.

Annamalai, P., and Lalithakumari, D. 1990. Decreasedsensitivity of field isolates toedifenphos. ology (4) : 553-558.

Annamalai, P. and Lalithakumari, D. 1992. Development ofresistance in to edifenphos -monotoring and control strategies.

(4) : 349-353.

Arora, R.K. 1994. Aggressiveness of metalaxyl resistant andsensitive isolates of . In : :

(Eds.: Shekhawat, G.S. , PaulKhurana, S.M., Pandey, S.K. and Chandra, V.K.).Indian PotatoAssociation, Shimla, pp. 179-183.

Arora, R.K., Kamble, S.S and Gangawane, L.V. 1992.Resistance to metalaxyl in inNilgiri Hills of southern India.

: 8-9.

Basu Chaudhary, K.C. and Puttoo, B.L. 1984. Fungicideresistant strains of in Kashmir - aprediction.pp. 509-514.

Brent, K.J. 1995.? FRAC Monograph No. 1,

GCPF, Brussels, pp. 48.

Brent, K.J. and Hollomon, D.W. 2007.? FRAC

Monograph No.1 (2 edition), Crop Life International,Brussels, Belgium. pp.55.

Cohen, Y., Baider, A. and Cohen, B.H. 1995. Dimethomorphactivity against Oomycete fungal pathogens.

: 155-1506.

Davidse, L.C. 1987. Resistance to acylalanines inin Netherlands.

: 129.

Davidse, L.C., Looijen, D., Turkensteen, I.J. and Van der Wal,D. 1981. Occurrence of metalaxyl-resistance strains of

in Dutch potato fields.: 65-68.

Dekker, J. 1985. The development of resistance to fungicides.

: 165-218.

Gangawane, L.V. and Reddy, B.R.C. 1985. Resistance of

to certain fungicides.: 23.

Gangawane, L.V., Kamble, S.S. and Arora, R.K. 1995.Synergistic effect of other fungicides on metalaxylresistant isolates of from NilgiriHills. : 159-162.

Gisi, U. and Staehle-Csech, U. 1989. Resistance riskevaluation of new candidates for disease control. In :

(Ed.: Delp, C.J.).APS Press, pp. 101-106.

Gupta, V.K. and Gupta, A.K. 1996. Use of forecasting systemin rational use of fungicides for apple scab control. In:

Eds.: Sokhi, S.S. and Thind, T.S.). NationalAgricultural Technology, Information Centre,Ludhiana, pp. 121-131.

Kaur, R., Thind, T.S. and Goswami, S. 2010. Profiling ofpopulations for metalaxyl

resistance and its management with novel actionfungicides.

(1) : 14-21.

Kumar, S., and Thind, T.S. 1992. Reduced sensitivity into carbendazim in

Punjab. : 103-105.

Lalithakumari, D., and Kumari, D.S. 1987. Screening forfield resistance in to edifenphos.

ology (3) : 342-347.

Lalithakumari, D. and Mathivanan, K. 1990. Strategies toavoid or delay or break resistance into edifenphos. (2) : 155-160.

Mohan, C., and Thind, T.S. 1995. Development ofca rbenda zi m re s i s t anc e i n

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