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Methods of molecular identifications and lab. protocols

Posted by Hetong Yang on 2008-05-09

Toxicity of Fungicides and Selective Medium Development for Isolation and Enumeration of Trichoderma spp. in Agricultural soils

Yang Hetong1,2,3, Maarten Ryder1, Tang Wenhua2

1: CSIRO Land and Water, PMB 2, Glen Osmond, SA 5064

2: China Agricultural University, Beijing, P.R. China. 100094

3: Biology Research Institute of Shandong Academy of Science, Jinan, P. R. China, 250014

Abstract

A Trichoderma selective medium was modified further by adding Baytan to stimulate Trichoderma conidiation, benomyl to promote vegetative growth of Trichoderma, and Triton X-100 to limit the colony size. Baytan was effective in suppressing Verticillium spp. The modified medium inhibited Penicillium and Aspergillus growth. Toxicity of various fungicides to Trichoderma and associated non-Trichoderma fungi was investigated to assist the development of a more reliable Trichoderma selective medium. The new medium is suitable for quantitative isolation of Trichoderma spp. from samples with high counts of non-Trichoderma fungi or low counts of Trichoderma spp. The quantification and isolation of Trichoderma spp. from soils with both high and low populations is possible.

Key words: Trichoderma; Selective medium; Fungicide; Quantification; Isolation

Introduction

Elad et al. (1981) developed a Trichoderma-selective medium (TSM), in which PCNB (pentachloronitrobenzene), Dexon (fenaminosulf, p-dimethyl-aminobenzenediazo sodium sulfonate; for suppression of Oomycetes), and rose-bengal were used as selective fungal inhibitors to enable the quantitative isolation of Trichoderma spp. from soils[1]. Later, it was found that if benomyl and was added to TSM, Fusarium spp. could be efficiently isolated from the soil[2]. Captan was added to TSM to eradicate Fusarium spp. when isolating Trichoderma spp. Askew and Laing[3] amended TSM with added metalaxyl or propamocarb-hydrochloride instead of fenaminosulf. Metalaxyl ws found to suppress the growth of Oomycetes, and propamocarb-hydrochloride reduced Aspergillus spp. and Penicillium spp. growth. Various TSM have also been developed by other groups[4].

We have used previously described the use of TSM [2,4] to quantitatively isolate Trichoderma spp. from various soil samples, including a Rhizoctonia-suppressive soil in South Australia[5], however these media proved to be unsuitable. This research aimed to increase the effectiveness of TSM for use in this and other soils. We investigated toxicity of a number of fungicides toward Trichoderma spp. and related fungi which confuse selective isolation from this soil. The work provides useful data for the selection of fungicides for use in selective media for isolation of Trichoderma. Based on preliminary observations in both the current study and previously[2,3], addition of TSM with fungicides was examined to (1) stimulate conidiation of Trichoderma spp., (2) stimulate growth of Trichoderma spp., (3) suppress Aspergillus, Penicillium and Verticillium.

Materials and Methods

Microbial strains

Trichoderma spp. T8, Sxb (isolated from wheat rhizosphere, Beijing, P. R. China); Trichoderma viride T1 isolated from wheat rhizosphere, Beijing, P. R. China); LTR-2 (isolated from a dry fruiting body of Ganoderma spp.), LR (a fast-growing mutant of LTR-2); Trichoderma harzianum T9, T22 (isolated from wheat rhizosphere, Beijing, P. R. China); Tc (isolated from cotton rhizosphere, Beijing, P. R. China); Trichoderma koningii Tk7a (isolated from an acid wheat field soil by A. Simon[5]; Gliocladium roseum GLR (from Prof. J. P. Tewari of Alberta University, Canada); Trichoderma pseudokoningii A5MH ; Trichoderma parceramosum A9-1 (isolated from Rhizoctonia-suppressive soil of wheat field, South Australia)[6]; Verticillium dahliae CV-1 (from diseased cotton, Jinan, Shandong, P. R. China).

Chemicals

Twenty six different fungicides were used in this study (Table 1). Fungicides were dissolved or suspended in 70% ethanol as stocks. Except where specified in the text, all fungicides were mixed into the media after autoclaving.

Table 1 Trade names and source of chemicals used in this study

Name

Formulation*

Supplier or producer

carbendazim, thiophanate-methyl, benomyl, curzate, kocide, dithane, acrobat, mancozeb, copper sandoz

CP

Agrochemical Supervision Institute, Ministry of Agriculture, P. R. China

Captan

80%WG

Crop Care, Australia

Fenaminosulf

60%WP

Dandong Agrochemical Plant, Liaoning Province, P. R. China

Asomate

CP

Heibei Guanlong Agrochemical Company, Ltd., P. R. China

Quintozene

CP

Linfen Chemical Engineering Factory, Shanxi Province, P. R. China

Metalaxyl

25%WP

Nantong Dye Chemical, Jiangsu Province, P. R. China

Iprodione

40%WP

Ronaplanc, France

Jinggangmycin

20%WP

Tonglu Huifeng Biochemical Co., Zhejiang Province, P. R. China

Tricyclazole

CP

Wenzhou Agrochemical Plant, Zhejiang Province, P. R. China

Rifampicin

CP

Xinchang Agrochemical Company, Zhejiang Province, P. R. China

Chlorothalonil

CP

Xingtai Agrochemical Plant, Hebei Province

Dithofencarb

CP

Xinyi Agrochemical Plant, Jiangsu Province, P. R. China

Polyoxin

CP

Yanbian Agrochemical Plant, Jilin Province, P. R. China

Triadimefon

CP

Yixing Biochemical Plant, Jiangsu Province, P. R. China

thiram

CP

Zanfeng Agrochemical Plant, Hebei Province, P. R. China

dimethachlon

CP

Zhejiang Chemical Engineering Academy

procymidone

CP

Zouping Agrochemical Plant, Shandong Province, P. R. China

* CP =Chemical (pure), WP = Wettable powder, WG = Wettable granule

Selective media

The final constituents of the media are shown in Table 2. TSB with the addition of reduced amounts of captan (0.04g/L, TSBcap0.04) and without captan addition (TSBcap-) were also used in the investigation. Chloramphenicol, streptomycin, Baytan, benomyl and captan were added to the media as 70% ethanol stocks after autoclaving. All the other ingredients were mixed together prior to autoclaving. The pH was not adjusted at any time (final pH 6.0-6.2). TSMC[4] and TSMAs[3] were included as reference media.

Table 2 Constituents of various selective media for the isolation and enumeration of Trichoderma spp.

Constituents

TSMC*

TSMAs*

TSB*

Glucose

3g

3g

3g

NH4NO3

1g

1g

1g

KH2PO4

0.9g

0.9g

0.9g

MgSO4

0.2g

0.2g

0.2g

KCl

0.15g

0.15g

0.15g

ZnSO4

0.02g

0g

0

FeSO4

0.02g

0g

0

MnSO4

0.02g

0g

0

Rose bengal

0.15g

0.15g

0.15g

chloramphenicol

0.25g

0.25g

0.25g

streptomycin

0.05g

0g

0.05g

PCNB

0.15g

0.15g

0.15g

metalaxyl

0.0125g

0.08g

0.08g

Baytan

0

0

0.08g

benomyl

0

0

0.0005g

captan*

0

0.1

0.1

Triton X-100

0

0

1ml

Agar

18g

18g

18g

Water

1000ml

1000ml

1000ml

*TSMC[4]

TSMAs[2,3].

Bioassay

Preliminary assay

Two wells (6 mm diameter) were dug at the opposite edges of PDA plates, which had been mixed in advance with fungal spores or other fungal propagules. Chemical suspensions or solutions (0.1 ml containing 10 mg/g) were poured into the wells. Inhibition zones were measured when the test fungi reached full growth after incubation for 4 d. Four replicates were included for each fungicide.

Fungal toxicity test

PDA (potato dextrose agar) mixed with chemicals at different concentrations was poured into Petri dishes. After solidification, an agar disc (diameter 6 mm) with fresh fungal mycelia (24 h) was placed in the center. Plates were incubated at 28C and when fungus on the nil plate had reached full growth, the diameter of the colonies was measured in two directions and the average was calculated. There were four replicates for each fungicide-fungus combination.

Statistics

The % Growth Inhibition (%) was calculated using the following equation:

Growth Inhibition (%) = (Do-Dx) /(Do-6)?100, where, Dx = Colony diameter on PDA with chemical amendments, and Do = colony diameter on PDA without chemical amendments.

Growth inhibition was converted into a probability (Y), concentrations of chemicals were converted to logarithmic values, and the least square equation (Y = a + bX) was obtained from Microsoft Excel. Middle inhibitory concentration (IC50) was calculated using the equation. Concentrations at which the growth of colony was completely inhibited were determined and designated as least complete inhibition concentration (LCIC).Six (6 in the formula) is the inoculant disc diameter.

Effect of chemicals on growth of Trichoderma spp.

Carbendazim or benomyl were added to PDA at different concentrations, and Trichoderma spp. were inoculated as an agar disc (6mm diameter) at the centre of plate. Plates were incubated at 28C. There were five replicates for each isolate-concentration combination, and colony diameter was measured when any of the plates was nearly covered by the fungal growth.

Effect of Baytan on conidiation of Trichoderma spp.

The stimulating effect of Baytan on conidiation of T. harzianum T9, T. koningii Tk7a, and T. pseudokoningii A5MH was tested. Each Petri dish contained 20 ml of PDA with different concentrations of Baytan. An agar disc carrying fresh mycelia of test fungi was inoculated at the center. The plate was incubated at 25C for 14 d to allow full conidiation, and each treatment was replicated 3 times. At the end of the incubation, the agar and mycelia/conidia were harvested into a flask containing 30ml sterilised saline. The flask was shaken vigorously to obtain a well dispersed suspension of conidia, which were enumerated microscopically with a haemocytometer.

Efficiency of selective media in the isolation of Trichoderma spp. S

oil samples were taken from a wheat field soils in which a general suppressiveness to Rhizoctonia solani had developed (Avon, South Australia[5]), and from field soils planted with potato, grape, parsnip, cabbage and onion (from Virginia, South Australia). After collection, the soils were kept sealed in polyethylene bags in a cold room. Depending on the dilution required, 1 to 50g of soil samples were assayed. Samples were diluted with autoclaved saline and the suspension was shaken vigorously on a vortex mixer for 60s before plating. Suspensions (0.1 ml) were spread onto plates which were incubated at 28C. After 7 d incubation, colony numbers and types were investigated. Trichoderma were identified to species level using macroscopic and microscopic features as described[8,9,10,11,12].

Results

Influence of chemical fungicides against Trichoderma spp. and V. dahliae

Among all 25 tested chemical fungicides (Captan was not tested), only 11 inhibited the growth of T. viride LTR-2 visually on a Petri dish at a concentration of 10 mg/g or less, in the preliminary assay. Further tests on the inhibitory effect of the 11 chemicals on LTR-2 are shown in Table 3. It was seen that carbendazim, benomyl and thiophanate-methyl were the most toxic to LTR-2, their EC50's being 0.7395, 1.6784 and 0.7370 mg/g respectively. Of the 11 chemicals tested, curzate showed the least toxicity to LTR-2 with an EC50 of 8295ug/g. Thiram and asomate gave EC50 values of 38.9214 and 44.2241 mg/g respectively. This result suggested that a number of fungicides could be adopted as selective agents without affecting growth of Trichoderma spp. However, only 6 fungicides showed high toxicity against V. dahliae CV-1 (Table 3). Thus, the selective fungicides were limited to those which reduced the counts of other non-Trichoderma fungi when Trichoderma spp. were the main target microbe for positive selection.

Table 3 Toxicity of selected fungicides to Trichoderma viride LTR-2 and Verticillium dahliae CV-1

Chemicals

T. viride LTR-2

EC50(mg/g)

V. dahliae CV-1

EC50(mg/g)

Carbendazim

0.7395

0.1103

Benomyl

1.6784

0.4673

thiophanate-methyl

0.7370

1.4248

PCNB

204.2597

1541.2135

dimethachlon

71.1640

n.d.

Asomate

38.9214

n.d.

Thiram

44.2241

n.d.

Kocide

128.7327

n.d.

curzate

8295.1631

n.d.

acrobat

437.2437

n.d.

tricyclazole

74.8469

n.d.

iprodione

n.d.

683.3014

Baytan

n.d.

1.4590

n.d. = not determined. R2 values ranged from 0.8491 to 0.9902)

Toxicity of carbendazim and benomyl to Trichoderma spp.

Carbendazim was generally the most toxic chemical fungicide toward Trichoderma spp., but different strains of Trichoderma spp. showed different sensitivities toward carbendazim. From Table 4, it is clear that Sxb, T9, T8 and T1 were more tolerant to carbendazim than LTR-2, especially T9 which showed an EC50 of 1008 mg/g. Isolates Tc, T22 and LR were more sensitive than LTR-2 to carbendazim, and LR was the most sensitive isolate, having an EC50 of 0.0438 mg/g.

The tolerance of Trichoderma spp. to benomyl differed from their tolerance to carbendazim. Isolate T9 showed the greatest tolerance to both fungicides, LTR-2 also showed some tolerance and the remaining strains were the most sensitive. LR was the most sensitive strain with an EC50 of 0.0015 mg/g. Other strains had similar EC50 values. Most wild isolates of Trichoderma spp. were slightly tolerant to both carbendazim and benomyl, compared to LR, a mutant of isolate LTR-2.

Table 4 Toxicity of carbendazim and benomyl to different Trichoderma isolates

Fungicide

Trichoderma spp.

EC50(mg/g)

LTR-2

0.5102

Sxb

3.0280

LR

0.0438

carbendazim

T22

0.3427

T9

1008.6942

T8

9.5760

T1

8.2713

Tc

0.5039

GLR

0.8000

Tk7a

0.1341

LTR-2

1.5113

Sxb

0.3122

LR

0.0015

benomyl

T22

0.3453

T9

7.3375

Tc

0.6191

Tk7a

0.5003

GLR

0.3330

Least complete inhibitory concentration (LCIC) of chemicals to isolates LTR-2 and CV-1

Carbendazim and benomyl were the most toxic chemical fungicides fungi other than Trichoderma, such as Verticillium, Fusarium, Penicillium, Aspergillus, Alternaria, Rhizoctonia, and Gliocladium (data not shown). An in vitro Petri dish test showed that at concentrations of 10-100 mg/g, carbendazim completely stopped the growth of most Trichoderma and Gliocladium spp.. Table 5 shows the lowest concentrations of carbendazim and other chemicals that completely inhibit the growth of Trichoderma spp. Carbendazim, benomyl, thiophanate-methyl, and PCNB showed LCICs to Verticillium dahliae of 0.7, 2.3, 2.6 and 2420 mg/g respectively, and to LTR-2 of 1.2, 3.4, 6.4 and 42602 ug/g, respectively. The results showed that apart from dimethachlon, all the other four fungicides listed in Table 5 had different LCICs, and all of them were more toxic to CV-1 than to LTR-2, indicating their possibilities as selective agents.

Table 5 Least complete inhibitory concentration (LCIC)of fungicides to T. viride LTR-2 and V. dahliae CV-1

Fungicide

CV-1, mg/g

LTR-2, mg/g

carbendazim

0.7

1.2

benomyl

2.3

3.4

thiophanate-methyl

2.6

6.4

dimethachlon

3780

820

PCNB

2420

42602

The stimulating effect of chemicals on the growth of Trichoderma spp.

It was found that when used at a relatively low concentration, several fungicides stimulated the growth of Trichoderma spp, even though at high concentrations they would be inhibitory. Table 6 shows that at concentrations < 3.2 mg/g, carbendazim stimulated the growth of T1, T8, and T9. Benomyl at < 0.4 mg/g showed stronger stimulating effect on LTR-2. The isolates tested were slightly more tolerant to these two fungicides than to others, but as shown in Table 4, a low concentration of either benomyl or carbendazim would not have an obvious adverse effect against those relatively sensitive isolates.

Table 6 Effect of different concentrations of carbendazim and benomyl on the growth (colony diam.; mm) of Trichoderma spp.

Fungicide

mg/g

0.00

0.50

0.10

0.20

0.40

0.80

1.60

3.20

T1

31.17A

ND

31.83B

32.5B

32.83B

34.67B

33.83B

29.33C

carbendazim

T8

27.33A

ND

31.33B

33.17B

33.67B

35.00B

31.00B

27.83A

T9

29.50A

ND

37.67B

38.17B

36.67B

36.50B

30.83B

28.17A

T1

31.55A

32.74A

34.26B

36.33B

34.47B

ND

ND

ND

benomyl

T8

27.42A

32.15B

36.05B

35.24B

35.53B

ND

ND

ND

T9

29.26A

40.78B

51.00B

52.78B

41.15B

ND

ND

ND

LTR-2

58.50A

74.50B

83.80B

81.30B

75.00B

ND

ND

ND

Note: Data followed by same letters in the same row were not significantly different (LSD0.05). ND-Not determined.

Influence of Baytan on conidiation of Trichoderma spp.

Baytan enhanced conidia production in the Trichoderma strains tested (Table 7). This effect made the colonies more easily discernible macroscopically.

Table 7 Stimulating effect of Baytan on conidiation (?108conidia/ml PDA) of Trichoderma spp.

Baytan, mg/g

A5MH

T9

Tk7a

Average

Increase %

Average

Increase %

Average

Increase %

0

0.86A

/

3.88A

/

0.05A

/

5

1.12B

30.2

5.93B

52.8

0.12B

140

10

1.16B

34.9

6.60B

70.1

0.11B

120

20

1.20B

39.5

5.71B

47.2

0.12B

140

40

1.15B

33.7

5.90B

52.1

0.11B

120

80

1.14B

32.6

5.24B

35.1

0.11B

120

Note: Data followed by same letters in the same row were not significantly different (LSD0.05).

Efficiency of selective media in the isolation of Trichoderma spp.

Results are shown in Table 8. Trichoderma growing on any of the test media produced flat colonies with mycelia of white and wet appearance. The reverse side of the colony was generally only slightly stained with Rose bengal. Old colonies exhibited a red centre when viewed on the reverse side. Green conidia, which appeared after 4-5 days' incubation, made identification much easier.

TSB containing 0.04g/L of captan performed the best as a selective medium when used to quantitatively isolate Trichoderma spp. from 12 soil samples. TSB (full recipe) and TSMA allowed the growth of the lowest numbers of Trichoderma spp., indicating that the selective agents would be possibly harmful to some strains or some species of Trichoderma genus. When TSB was used without captan, i.e. TSBcap-, greater numbers of Trichoderma spp. were recorded. TSMC showed the similar results to TSBcap-. The results indicated that the high concentration of captan was responsible for the reduced number of Trichoderma spp. Baytan did not reduce counts of Trichoderma spp., but stimulated conidiation.

Non-Trichoderma fungi which appeared on TSB0.04 and TSBcap- media were mainly Penicillium spp. and Aspergillus spp., but no Verticillium. On TSMC and TSMAs, Verticillium spp. appeared occasionally, which would confuse the microscopic identification of Trichoderma spp. TSB with 0.1g/l captan addition showed almost no counts of non-Trichoderma fungi, whereas TSMAs showed a certain amount of non-Trichoderma fungi, indicating the suppressive effect of benomyl and Baytan to these non-Trichoderma fungi. TSBcap- and TSMC gave much higher counts of non-Trichoderma fungi, indicating their limited feasibility when used for samples with high populations of non-Trichoderma fungi.

Table 8 Recovery of Trichoderma spp. (cfu/g dry soil) from various soil samples using different selective media

Field soil TSMC TSMAs TSB TSB(0.04) TSBcap-
TR

NTR

TR

NTR

TR

NTR

TR

NTR

TR

NTR

Potato

442AB

2153A

59A

74B

605BC

0B

973C

0B

1475D

5649C

Cabbage

83AB

1239A

33A

72B

94B

0B

78AB

6B

161C

2572C

Onion

28AB

4274A

6A

11B

0A

0B

44B

17B

6A

3009C

Parsnip

0A

2667A

0A

19B

0A

0B

14B

19B

0A

2474A

Grape

22A

2119A

0A

0B

0A

0B

11A

0B

123B

5825C

Wheat

0A

2874A

0A

4B

0A

0B

12B

1714B

0A

5143D

Wheat

4A

2443A

0A

23B

0A

0B

4A

408B

0

posted by Hetong Yang on 09 May, 2008

Copyright: Irina Druzhinina & Alexey Kopchinskiy 2004 - 2008