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Screening of Fungicide to Control Ciboria carunculoides Under Laboratory Conditions.

Byline: Mingqiang Ye, Zheshi Kuang, Xiangjie Zhao, Qiong Yang, Qingrong Li, Yang Xiao, Zhenjiang Wang, Fanwei Dai and Guoqing Luo

Abstract Ciboria carunculoides is one of the major fungal pathogen that attack mulberry grown worldwide as a crop for silkworm rearing. The use of chemical fungicides is one of the main tactics being used for the management of plant diseases.

The effect of 15 fungicides on radial growth of C. carunculoides was studied in order to screen out fungicides which are highly effective against this fungus. Carbendazim was proved to the most aggressive showing the lowest EC50 value (8.04 mg/l), and glyphosate was the least virulent with a mean EC50 value of 8741.78 mg/l.

The mixtures of fungicides at different concentration with mulberry juice were tested for C. carunculoides growth inhibition, and the results showed that Carbendazim + juice mixture was most aggressive having the lowest EC50 value (521.01 mg/l), when compared with Procymidone + juice mixture.

The percent conidial germination of C. carunculoides was significantly affected by different concentration of Carbendazim and Glyphosate used at different concentrations in soil.

Both the fungicides used in this study caused 100% inhibition of fungal germination at the concentration of 40 and 80 mg/l, respectively. Our study has helped to screen some highly effective fungicides for C. carunculoides management.

Keywords: Fungicide, Ciboria carunculoides, carbendazim, glyphosate.

INTRODUCTION

Mulberry is grown worldwide as a crop for silkworm rearing. Apart from this many parts of mulberry tree have been used as uncooked or processed foods for health care (Hong et al., 2007). Mulberry fruits also contain several substances with medical action.

Due to increase in fruit demand, growing area of the tree has remarkably increased (Kishi, 1998). According to the index of plant diseases, mulberries are susceptible to many pathogens (Anonymous, 1960). Ciboria carunculoides (Siegler and Jenkins) Whetzel, C. shiraiana (Henn.) Whetzel and Scleromitrula shiraiana (Henn.) Imai have been reported separately as causal agents of the fruit diseases (Kishi,

1998; Kohn and Nagasawa, 1984; Whetzel and Wolf,

1945).

Ciboria carunculoides is one of the major fungal pathogens that attack mulberry fruits by causing disease named as "popcorn disease". Popcorn diseased mulberry fruits have greatly enlarged ovaries with small and succulent calyx

lobes in comparison with fleshy lobes of normal fruits (Gray and Gray, 1987).

Diseased mulberry fruits have ovaries filled with fungal mycelium rather than having normal plant tissues. The pathogen releases vast number of fungal spores, resembling a waxy substance on outside ovary wall. As a result, normal drupelets formation and maturation are prevented, thereby destroying the mulberries as edible fruit (Siegler and Jenkins,

1922).

The use of chemical fungicides is one of the main tactics being used for the management of plant diseases. Knowledge is scarce about the management of C. carunculoides by using chemical fungicides. Chai et al. (2005) showed that 25% Amistar SC was more effective on popcorn disease when compared with conventional pesticides carbendazim and thiophanate-methyl under field conditions.

Therefore Amistar could be applied in production to control popcorn disease of mulberry. The aim of the present work was to test the impact of different fungicides on growth inhibition, sporulation and other biological parameters of C. carunculoides which can provide the basic information for future management of C. carunculoides under field conditions.

MATERIALS AND METHODS

Fungal strains

Ciboria carunculoides isolate, Cc01,

originally isolated from Mulberry, deposited at the

collection at Sericulture and Agri-food Research

Institute of Guangdong Academy of Agricultural Sciences, identified as Hu et al. (2011) by PCR, was used in this study.

To produce the inoculum for each assay, C. carunculoides was cultured on potato dextrose agar (Potato infusion 200 g/L; Dextrose 20 g/L and Agar 20 g/L) and incubated at 20+-2degC for

10 days. Conidia were harvested with distilled water containing 0.03% Tween-80, (Whiga chemicals Shanghai). Conidia were counted in a Fuchs-Rosenthal hemocytometer and a suspension of 1x107 conidia/ml was prepared.

Fungicides

List of pesticides used in the study, molecular formula, manufacturer/supplier are given in Table I whereas the concentrations of different fungicides used in this study are given in Table II.

Effects of different fungicides

Radial growth

Potato dextrose agar (PDA) was sterilized at

121degC at 15 psi for 25 minutes.

Freshly prepared fungal suspension (100 ul of 1 x 106 conidia ml-1) was inoculated in the centre of plates using a micro applicator and was spread to cover the whole plate. The plates were incubated at 20 +- 2 degC, 80 +- 5% R.H., and L14:D10 h for 2-3 days.

Mycelial discs together with medium (O1 cm) were removed and

cultured on PDA having the different concentrations of fungicides (Tables II, III), respectively, and incubated at 20 +- 2oC.

The same medium without fungicides served as a control. There were 10 Petri dishes for each concentration of different fungicides. Colony diameters were measured after 4-5 days. All the treatments were replicated three times.

The average diameter of every colony was calculated as (long diameter + short diameter) / 2 (Ali et al.,

2009). Inhibitory percentage on mycelia growth was calculated as:

% Inhibition of mycelial growth = [(Mc-Mt)/Mc]100

where Mc=Dimeter of mycelium in control and

Mt=Diameter of mycelium in treatment.

Table I.- Fungicides used in the study.

Fungicide (crude) Molecular formula Manufacturer/

supplier

Tebuconazole C16H22ClN3O Bayer

Nicotinamide C6H6N2O BASF

Metiram C12H12N6S16Zn BASF

Thiophanate-Methyl C12H14N4O4S2 BEST

Carbendazim C2H5NO2 Jiangshu

Nanfong Co.

Iprodione C13H13Cl2N3O3 Bayer

Dimetachlone C10H7Cl2NO2 Zhejian

Heyipest.Co.

Iprovalicarb C18H28N2O3 BEST

Zineb C4H6N2S4Zn Shenzhen

Noposion

Prochloraz C15H16Cl3N3O2 Jiangshu

Huifong Co.

Procymidone C13H11Cl2NO2 Sumitomo

Chemical

Triadimefon C14H16ClN3O2 Jiangshu

Qizhounushe

Chemical

Difenoconazole C19H17Cl2N3O3 Shandong United

Pesticide

Industry Co.

Glyphosate C3H8NO5P Monsanto

Company

Table II.- Fungicides and concentrations used in this study.

Fungicides Concentrations (mg/l)

Carbendazim 5 10 20 40 80

Iprodione 300 600 1200 2400 4800

Prochloraz 15.63 31.25 62.5 125 250

Dimetachlone 25 50 100 200 400

Procymidone 35 70 140 280 560

Glyphosate 3380 6750 13500 27000 54000

The curves of (log concentration - probit line (LC-p)) were calculated and tested by chi-square test, median lethal concentrations (LC50) and their confidence intervals were calculated by probit analysis using SPSS (Statistical Package for Social

Science) 8.0 for windows (SPSS, 1997).

Germination of C. carunculoides

Six different fungicides were selected based on previous section (2.3). Liquid culture medium was prepared with KNO3 10 g, KH2PO4 5 g, MgSO4

2.5 g, FeCl3 0.02 g, Sucrose 50 g dissolved in

deionized water 1000 ml and then the medium was

sterilized at 121degC at 15 psi for 25 minutes.

Different fungicides were added to the basal medium at three different concentrations (according to the EC95 values of experiment 2.4, Table V) while the basal medium without any fungicide served as control. Ciboria carunculoides (1x104 conidia/ml) was added to each flask.

The flasks were incubated at

20+-2degC. Percent germination was calculated after

36-48 h of shaking at 200 rpm by placing one ml of suspension on cavity slides.

Three separate fields

were observed for germination at 400 X magnifications for each treatment and 100 conidia were observed randomly in each field. Conidia with

germ tubes equal to or greater than the width were

considered to have germinated.

The entire experiment was replicated three times on different dates.

Effect of different fungicides mixed with mulberry juice on radial growth of C. carunculoides

The effects of different mixtures at six different concentrations on radial growth were studied by using the method of 2.3. The freshly

prepared PDA medium having different

concentrations of fungicides (Table VI) was added to 9 cm petridish followed by covering a layer of mulberry leaf juice (300 ul, having 60 ug/ml Ampicillin).

Mycelial discs together with medium (O1 cm) were removed to each flask. The flasks were incubated at 20+-2oC. The same medium without the mixture of fungicide and juice served as a control.

The fresh mulberry leaf with flexible branch was washed with deionized water three times, and then was put into a small mill for fresh mulberry juice.

Effect of cabendazim and glyphosate against C. carunculoides in soil Three different concentrations of two fungicides (carbendazium and glyphosate) were selected based on experiment 2.4.

The soil collected from the vicinity of mulberry tree mixed with C. carunculoides (10 ml of 1 x 103 conidia ml--1) was put into a plastic box with (30x15x8cm). Different concentrations of fungicides (Table VII) were sprayed with 500ml sprayer to soil surface while the box without any fungicide served as control.

The plastic boxes were incubated at 202degC for 10 days. Inhibitory percentage on germination was calculated as:

% inhibition = [(Gc-Gt)/Gc]100

where Gc = germination in control and Gt =

germination in treatment.

The entire experiment was replicated three

times on different dates.

Statistical analysis

All experiments were carried out in triplicate and the results were expressed as average of triplicate determinations. Radial growth, inhibition growth and inhibition germination data were analyzed by Analysis of variance (ANOVA) and treatment means were compared by using Tukey's HSD test for mean comparisons at 5% level of significance. All statistical analysis was performed using SAS 8.01(2000).

RESULTS

Effect of different fungicides

Radial growth of C. carunculoides

The effect of 15 fungicides on radial growth of C. carunculoides was studied to screen out fungicides which are highly effective against this fungus.

The average radial growth of C. carunculoides was significantly affected by different fungicides. Maximum radial growth (1.3+-0.2 cm) was observed for Thiophanate-Methyl whereas no radial growth was observed for Tebuconazole, Nicotinamide, Prochloraz, Carbendazim, Dimetachlone, Difenoconazole and Glyphosate (Table III).

The fungicides of Carbendazim, Iprodione, Dimetachlone, Prochloraz, Procymidone and Glyphosate were used to study further on the basis of their economics and active mechanisms on controlling pathogen in crop field.

The half-maximal effective concentration (EC50) of different fungicides against C. carunculoides is shown in Table IV.

Carbendazim was proved to the most aggressive showing the lowest EC50 value (8.04 mg/l), and Glyphosate was the least virulent with a mean EC50 value of 8741.78 mg/l. Statistically similar EC50 values were obtained for Dimetachlone, Prochloraz and Procymidone (Table IV).

Table III.- Effect of different fungicides on radial growth of C. carunculoides (4d).

Concentration Radial growth

(g/L) (cm)

(Mean+- S.E)

Tebuconazole 8.6 0.0 +- 0.0 d

Nicotinamide 10 0.0 +- 0.0 d

Metiram 14 0.9 +- 0.0 ab

Thiophanate-Methyl 14 1.3 +- 0.2 a

Carbendazim 10 0.0 +- 0.0 d

Iprodione 10 0.3 +- 0.1 c

Dimetachlone 8 0.0 +- 0.0 d

Iprovalicarb 14 0.9 +- 0.2 ab

Zineb 13 1.1 +- 0.2 a

Mancozeb 16 0.7 +- 0.0 b

Prochloraz 5 0.0 +- 0.0 d

Procymidone 2 0.8 +- 0.1 b

Triadimefon 4 0.7 +- 0.1 b

Difenoconazole 2 0.0 +- 0.0 d

Glyphosate 80 0.0 +- 0.0 d

F =96.25, df=14,

PLess than0.0001

Means +- SE in the same column followed by different letters are significantly different (Tukey's HSD Test, PLess than0.05)

Germination of C. carunculoides

The percent conidial germination of C. carunculoides was significantly affected by different fungicides used at different concentrations (based on EC95 value of above experiment) as shown in table V. All the fungicides used in this study caused 100% inhibition of fungal germination at the highest concentrations whereas the germination inhibition differed at lower concentrations of different fungicides.

The highest germination inhibition (96.2%) at the lowest concentration was caused by carbendazim when used at 60 mg/l. The lowest germination inhibition (92.1%) at the lowest concentration was observed for Procymidone when used at 650 mg/l (Table V).

Effect of different fungicides mixed with mulberry juice

Radial growth of C. carunculoides

The effect of mixtures of fungicides at different concentration with mulberry juice on growth inhibition of C. carunculoides is shown in Table VI.

The average radial growth of C. carunculoides was significantly affected by different treatments. The highest growth inhibition (97.96%) among treatments was observed for mixture of carbendazim 4000 mg/l and mulberry juice whereas the lowest growth inhibition was observed for the mixture having Procymidone 625 mg/l+ mulberry Juice (Table VI).

The half-maximal effective concentration (EC50) of different fungicides and juice mixtures against C. carunculoides is shown in Table VII. Carbendazim + juice mixture was proved more aggressive showing the lowest EC50 value (521.01 mg/l), when compared with Procymidone + juice mixture with a mean EC50 value of 1322.55 mg/l (Table VII).

Effect of Cabendazim and Glyphosate against C. carunculoides in soil

The percent conidial germination of C. carunculoides was significantly affected by different concentration of Carbendazim and Glyphosate used at different concentrations in soil as shown in Table VIII.

Both the fungicides used in this study caused 100% inhibition of fungal germination at highest concentrations whereas the germination inhibition differed at lower concentrations of different fungicides. The lowest germination inhibition (82.1%) at lowest concentration was caused by carbendazim when used at 10 mg/l.

The lowest germination inhibition (80.7%) at lowest concentration was observed for glyphosate when used at 40 mg/l (Table VIII).

DISCUSSION

Progressive increase of production and application of chemicals fungicides for agriculture as well as for plant protection has converted the problem of environmental pollution into national and international issue. (Plimmer, 1990; Mathys,

1994).

Therefore, more efforts are being directed

Table IV.- Regression analysis of probit Inhibition growth and log-concentration data of bioassay with fungicides against C. carunculoides (4d).

Fungicide Regression equation (R2) EC50 (mg/l) (Mean+- S.E) EC95 (mg/l) (Mean+- S.E)

Carbendazim Y=3.2813+1.8987X (0.9862) 8.04 +- 0.8 59.09 +- 1.4

Iprodione Y=0.1824+1.5571X (0.9984) 1241.54 +- 11.3 14137.06 +- 20.6

Dimetachlone Y=1.5386+2.0265X(0.9950) 51.06 +- 1.2 330.93 +- 9.3

Prochloraz Y=2.2099+1.8054X(0.9908) 35.10 +- 2.1 286.05 +- 8.7

Procymidone Y=1.7241+1.7464X(0.9941) 75.12 +- 6.2 657.08 +- 9.2

Glyphosate Y=-4.2311+2.3420X (0.9819) 8741.78 +- 23.5 44051.32 +- 27.9

Table V.- Germination percentages of C. carunculoides conidia suspended in nutritional liquid containing varying concentration of different fungicides (36 - 48h, 20+-2oC).

Inhibition

Concentration

Fungicide germination % (Mean+-

(mg/l)

S.E)

Carbendazim 60 96.2 +- 1.2 b

100 100.0 +- 0.0 a

Iprodione 14500 92.7 +- 4.7 c

16000 99.5 +- 3.1 b

20000 100.00 +- 0.0 a

F=72.34, df=2, PLess than0.0001

Prochloraz 280 95.9 +- 2.9 c

300 98.5 +- 3.4 b

320 100.0 +- 0.0 a

F=65.28, df=2, PLess than0.001

Dimetachlone 350 94.1 +- 5.2 b

500 100.0 +- 0.0 a

600 100.0 +- 0.0 a

F=80.41, df=2, PLess than0.001

Procymidone 650 92.1 +- 5.2 b

800 100.0 +- 0.0 a

900 100.0 +- 0.0 a

F=92.13, df=2, PLess than0.0001

Glyphosate 45000 95.3 +- 4.7

55000 99.4 +- 1.8

65000 100.0 +- 0.0

F=63.70, df=2, PLess than0.001

using selective chemicals as well assessment and usage of their minimum concentration required for disease management (Wania and Mackay, 1996; Larson et al., 1997).

Initially, screening bioassays towards were carried out to determine most suitable fungicides for the management of mulberry popcorn disease on the basis of their effectiveness as well as

Table VI.- Effect of different mixtures at six different concentrations on radial growth of C.

carunculoides (4d).

Concentration Inhibition growth

Fungicide (mg/l) (%) (Mean+- S.E)

Carbendazim+Juice 250 32.65 +- 3.2 e

500 43.88+- 2.7 d

1000 66.33 +- 4.1 c

2000 85.31 +- 5.3 b

4000 97.96 +- 2.9 a

F=154.30, df=4,

PLess than0.0001

Procymidone + Juice 625 27.84 +- 2.2 e

1250 46.39 +- 3.1 d

2500 67.01 +- 4.5 c

5000 88.66 +- 1.2 b

10000 97.94 +- 3.5 a

F=127.45, df=4,

PLess than0.0001

economics. All the chemicals used in this study effectively reduced the radial growth of C. carunculoides. The concentrations of different fungicides were selected on the base of reduction in radial growth. In order to observe inhibition of spore germination and mycelial growth in subsequent experiments, EC50 and EC95 concentrations of fungicides were calculated.

EC50 and EC95 values of carbendazim (8.04 and 57.09 mg/l) calculated in this study were higher EC50 of carbendazim against Botryosphaeria berengriana (0.108 mg/l) observed by Li et al. (2009). This difference in EC50 values can be due to the possible nature of the pathogen.

Table VII.- Regression analysis of probit Inhibition growth and log-concentration data of bioassay with mixtures against C. carunculoides.

Fungicide Regression equation (R2) EC50 (mg/l) EC95 (mg/l)

Carbendazim + Juice Y=-0.5898+2.0574X (0.9803) 521.01 +- 9.4 3283.48 +- 27.4

Procymidone + Juice Y=-1.7990+2.1782X(0.9930) 1322.55 +- 21.5 7526.32 +- 36.7

Table VIII.- Effect of Cabendazim and Glyphosate against C. carunculoides in soil (10d).

Fungicide

(mg/l) (Mean+- S.E)

Carbendazim 10 82.1 +- 5.2 b

20 96.9 +- 4.9 a

40 100.0 +- 0.0 a

F=98.14, df=2, PLess than0.0001

Glyphosate 40 80.7 +- 6.3 b

60 97.5 +- 3.1 a

80 100.0 +- 0.0 a

F=102.36, df=2, PLess than0.0001

Means +- SE in the same column followed by different letters are significantly different (Tukey's HSD Test, PLess than0.05)

The disease management strategies are mainly pre-emergence and post emergence. Pre-emergence disease management is carried out at initial stage and contact poisons are used for such disease management whereas after plant growth and initial establishment of disease post emergence strategies are used with systemic fungicides (Galloway, 2008).

In this study, the purpose of using six fungicides including contact and systemic fungicides was to find out any possible way of controlling disease (Table V), at the same time, the EC95 concentrations of six fungicides were selected to test the maximum control efficacy. It is necessary to use systemic fungicides after disease infected plant internal organs and established in plant.

During these studies Carbendazim and Procymidone which are well known systemic fungicides were combined with mulberry juice for possible disease management after the establishment of C. carunculoides on mulberry branches and fruits. The average radial growth of C. carunculoides was affected by different treatments.

Highest growth inhibition (97.96%) was observed for mixture of carbendazim 4000 mg/l and mulberry juice whereas

lowest growth inhibition (27.84%) was observed for

the mixture having Procymidone 625 mg/l+

mulberry Juice showing that carbendazim is more

effective for management of this disease.

In order to imitate the effect of the internal environment of plant on fungicide, the project of fungicide and mulberry juice mixture was designed. High concentrations of two systemic fungicides were used to test the efficacy of Carbendazim and Procymidone based on the low efficacy at low concentration of these two fungicides in early experiment (Table VI).

In last part of our studies, efforts were made to imitate the field conditions by applying the C. carunculoides to the soil already collected from the vicinity of mulberry tree. After establishment of C. carunculoides, Carbendazim and Glyphosate were applied to this culture at different concentrations.

Both the fungicides used in this study caused 100% inhibition of fungal germination at the highest concentrations whereas the germination inhibition differed at lower concentrations of different fungicides showing the possibility of using these chemicals for C. carunculoides at initial stage or during dormant stage when the pathogen stays in soil for its growth and survival.

Our results have showed that the variation in effectiveness of different chemicals against C. carunculoides is dependent upon different unknown factors. Carbendazim was proved to be most effective chemical for C. carunculoides management at different stages of disease establishment. We hope that the findings of the current studies will provide basic information for C. carunculoides management in field conditions.

ACKNOWLEDGEMENTS

The research was funded by grants from the Modern Agro-industry Technology Research System (No.CARS-22).

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