Selective Medium for Isolation of Mycoleptodiscus terrestris ... - NCBI

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ation of M. terrestris from target and nontarget plants, sediments, and organic debris of aquatic environments. The selectivity of Martin's rose bengal agar (MRBA) ...
Vol. 56, No. 11

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3273-3277

0099-2240/90/113273-05$02.00/0 Copyright © 1990, American Society for Microbiology

Selective Medium for Isolation of Mycoleptodiscus terrestris from Soil Sediments of Aquatic Environments BARUCH SNEHt* AND JAMES STACK EcoScience Laboratories, Inc., 85 North Whitney Street, Amherst, Massachusetts 01002 Received 8 June 1990/Accepted 6 August 1990

A selective medium was developed for the dilution plate isolation of Mycoleptodiscus terrestris from natural soils and sediments from aquatic environments. The ingredients per liter of the selective medium are as follows: KH2PO4, 0.5 g; MgSO4- 71120, 0.5 g; dextrose, 10.0 g; peptone, 5.0 g; chloramphenicol, 0.25 g; rose bengal, 50 mg; oxgall, 5.0 g; Terraclor (pentachloronitrobenzene, 75% active ingredients), 0.5 g; agar, 15.0 g. After autoclaving, the following ingredients were aseptically added: sorbic acid (0.7% autoclave-sterilized aqueous solution), 5.0 ml; Subdue (25.1% emulsion of metalaxyl), 0.5 ml; Truban (40.7% suspension of etridiazol), 0.05 ml. The colony-restrictive properties of this medium enabled its use in the drop plate method, originally developed for viable counts of bacteria. Alfalfa sprouts as baits were not suitable for quantitative recovery of the fungus, although 5% of alfalfa sprouts were infected with M. terrestris when incubated on soil containing 1.5 x 102 CFU/g.

The fungus Mycoleptodiscus terrestris (Gerdemann) Ostazeski is being developed as a microbial control agent of the nuisance aquatic plant Eurasian watermilfoil, Myriophyllum spicatum L. (2; Y. Limpa-Amara, Ph.D. thesis, University of Massachusetts, Amherst, 1988). It is important to evaluate the fate and spread of M. terrestris after its introduction to an aquatic environment for the control of milfoil. A selective medium is therefore an important tool for the isolation, identification, and enumeration of M. terrestris from target and nontarget plants, sediments, and organic debris of aquatic environments. The selectivity of Martin's rose bengal agar (MRBA) (3), previously used to isolate this fungus (Limpa-Amara, Ph.D. thesis), was not satisfactory; colonies of M. terrestris expanded on the plates, making enumeration inaccurate and difficult. It allowed growth of a variety of soil and aquatic fungi, which in turn inhibited growth and/or masked the colonies of M. terrestris on the plates. This work describes the development of a selective medium for the isolation, identification, and enumeration of M. terrestris from natural soil and lake sediment.

5.0 mg; H3BO3, 5.6 mg; MnCl2 4H20, 3.7 mg; FeCl2 .4H20, 1.5 mg; NaMoO4. 2H20, 1.8 mg; CUSO4 5H20, 0.4 mg; agar, 15.0 g. (ii) Czapek agar. Czapek agar (Difco Manual) was used in some of the experiments. (iii) Modified Czapek agar. Pectin (from citrus; Sigma Chemical Co.), 5 g/liter, and/or carboxymethyl cellulose (Sigma), 5 g/liter, was added to Czapek agar to replace sucrose to evaluate their possible utility in the isolation of M. terrestris. (iv) MRBA. MRBA (3) was used as the standard isolation medium. It contained the following ingredients per liter: K2HPO4, 0.5 g; KH2PO4, 0.5 g; MgSO4 7H20, 0.5 g; dextrose, 10.0 g; peptone, 5.0 g; yeast extract, 0.5 g; rose bengal, 50 mg; agar, 15.0 g. Streptomycin sulfate (30 mg) was aseptically added to the medium after autoclave sterilization. (v) Basic medium. The basic medium (a modified MRBA) used in this study contained the following ingredients per liter: KH2PO4, 1.0 g; MgSO4 7H20, 0.5 g; dextrose, 10.0 g; peptone, 5.0 g; chloramphenicol, 0.25 g; rose bengal, 50 mg; agar, 15.0 g. Antimicrobial compounds. To determine the suitability and concentrations of antimicrobial ingredients for the selective medium, various compounds were added separately and in combination. Their effects on recovery and growth of M. terrestris as well as on restriction of undesired soil fungi (such as Aspergillus sp., Penicillium sp., unidentified phycomycetes, and others) on dilution plates were evaluated. The following compounds were tested: Benlate (50% benomyl; Du Pont, Wilmington, Del.); Banrot (25% 3-thioallophenate and 15% Truban; Sierra Chemicals, St. Louis, Mo.); Captan (50% active ingredients [a.i.], Chevron Corp., Richmond, Calif.); cinnamic acid; coumarin; Dodine (65% a.i.); gallic acid; Kocide 101 (75% Cu hydroxide; Griffin Corp., Valdosta, Ga.); oxgall; Subdue (25.1% metalaxyl; Ciba Geigy, Basel, Switzerland); tannic acid; Terraclor (75% pentachloronitrobenzene; Uniroyal, Middlebury, Conn.); and Truban (40.7% etridiazol; Sierra Chemicals). Procedures. Hyphae of M. terrestris grown in potato dextrose salts broth were fragmented in 0.03 M phosphate buffer, pH 7.0, in a Waring blender for 1 min and then concentrated and stored at 4°C until used. The suspension -

-

MATERIALS AND METHODS Fungal isolates. The isolate of M. terrestris used in this study was described previously (1, 2; Limpa-Amara, Ph.D. thesis). Aspergillus sp., Penicillium sp., and unidentified phycomycetous fungi isolated from lake sediment by using soil dilution plates on MRBA were used in preliminary stages of testing the effects of the various fungicides on M. terrestris and the most common soil fungi that interfere with isolation and identification of M. terrestris on MRBA. Media. (i) Potato dextrose salts agar or broth. Potato dextrose salts agar or broth was used in most experiments for growth of M. terrestris. These media contained the following ingredients per liter: potato starch, 10.0 g; yeast extract, 2.0 g; dextrose, 10.0 g; MgSO4- 7H20, 0.2 g; ZnSO4 7H20, 7.3 mg; CoCl2 6H20, 6.0 mg; CaCl2 .2H20, Corresponding author. t Permanent address: Department of Botany and Institute for Nature Conservation Research, Tel Aviv University, Ramat Aviv, *

Israel 69978. 3273

APPL. ENVIRON. MICROBIOL.

SNEH AND STACK

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TABLE 1. Effect of addition of different fungicides to the basic medium on growth of M. terrestris and on inhibition of other fungi from soil sediment Concn

tris linear

(,ug/ml)

growth

Linear growth

100

100

100

10 20 30 40 50 100

87

95

93 83

84 63

90 90

51

86

0

1

2,500 5,000

150 150

50 100 250 500 10 50 100 250 10 50 100 250

75 75 75 75 90

113 53 97

97 83

53 44 35 35 30 18 67 33 80 80 67 83

Oxgall Terraclor

Subdue

Truban

Subdue + Truban Dodine

73 80 100 57 117 113 87 41 0 77 96 73 0 83 71 0 109 91 77 98 92 76

83 133 117 50 67 83 80 30 0 100 79 79 0 88 93 0 114 72 72 57 43 57

Banrot Kocide Captan

Benlate Tannic acid Gallic acid Coumarin Cinnamic acid

150 + 20 10 100 1,000 1 10 50 10 >50 1 10 100 1,000 10 100 1,000 10 100 1,000 10 100 1,000

recov-

110

0

6b 75 62b

3lb

Presented as a percentage of growth on the basic medium with fungicides after 5 days at 27°C, as described in Materials and Methods. b Browning around the colony, strong inhibition. a

Growthc

Recov-

Growthc

lood 26 9

io0f 57 39

++++ +++ +

looe 100 92

+++ +++ +++

++ + +

59 76 64

+++-

a Hyphal fragments (grown in liquid culture) were mixed into the soil dilutions (about 60 propagules per ml of suspension). b The selective medium contained the following fungicides per liter: oxgall, 5 g; Terraclor, 0.5 g; sorbic acid, 35 mg; Subdue, 0.5 ml; Truban, 0.05 ml. c Relative colony diameter on the media in different treatment, where + is 10 mm. d Growth of M. terrestris without soil suspension on basic medium was used as the control for its growth on basic medium after soil suspension. e Growth of soil fungi from soil suspension on the basic medium was used as the control for their growth on both the basic and the selective media. f Growth of M. terrestris without soil suspension on MtSA was used as the control for its growth from soil suspension on MtSA.

83 57 79 45 23

0 69 50 0 75 47b

Basic medium M. terrestris (Mt) Soil, 5 x 10-3 Mt + soil, 2 x 1i-0 Mt + soil, 5 x 10-3

Selective medium (MtSA) M. terrestris Soil, 5 x 10-3 Mt + soil, 2 x 1(-3 Mt + soil, 5 x 10-3

90

87 150 150 0 17 5 0 0 0 0 62 6

Recov-

Soil fungi

ery ()ery(%

CFU

ereda

(%)a Basic medium Sorbic acid

%

M. terrestris

Medium and soil dilution

qnil avit fiinLyl iuiigi

M. terresTreatment

TABLE 2. Recovery of M. terrestrisa from sediment soil and inhibition of other fungi on the selective medium'

no

was mixed with sediment (taken from the bottom of a lake) for isolation and enumeration on dilution plates. The effects of the antimicrobial components on growth of M. terrestris were evaluated by using mycelia taken from margins of 3- to 5-day-old potato dextrose salts agar cultures, sclerotia taken from 2-week-old cultures, and hyphal fragments grown in potato dextrose salts broth and stored at 4°C, mixed with sediment soil dilution. Soil dilutions were made in 0.1% agar solution to stabilize the suspension. Aliquots of 0.1 ml of soil dilution were evenly spread on the agar surface with a sterile bent glass rod. After the plates were incubated at 28°C for 3 to 5 days, colony size was measured and numbers of colonies (both M. terrestris and other soil fungi) were recorded. Samples of 30 colonies

typical for M. terrestris were transferred from M. terrestris selective medium (MtSA) soil dilution plates to MRBA plates to verify positive identification of M. terrestris. Experiments with different fungicide concentrations were carried out in duplicate and repeated at least twice. Experiments with the final selective medium containing all antimicrobial components were carried out in four replicates and conducted three times. (It is the routine used in our laboratory.) Drop plate method (4). A cross, dividing the plate into four quarters, was marked on the bottoms of the plates. Five drops (10 [LI) from each of four 10-fold serial dilutions of M. terrestris suspensions were placed in each quarter. The plates were left open in the laminar flow hood until the drops were absorbed and dry. CFU were counted under a dissecting microscope (x 100) after 4 days of incubation at 28°C. Baiting procedure. Alfalfa seeds were surface sterilized in 3% sodium hypochlorite solution for 1 min, rinsed in sterile water, and placed on moist double-layer towel paper for germination. After 3 days of incubation at room temperature (12-h fluorescent light photoperiod), sprouts (60 per plate) were transferred to plates containing 10 g of soil and 10 ml of M. terrestris hyphal fragment suspension (15, 1.5 x 102, 1.5 x 103, and 1.5 x 104 CFU/g of soil) in four replicates. After 3, 5, and 7 days of incubation at room temperature (12-h fluorescent light photoperiod), 20 sprouts were recovered from each plate, surface sterilized in 1% sodium hypochlorite solution for 1 min, washed with sterile water, and blotted on towel paper. Ten sprouts per plate were placed on the new selective agar. After 5 days of incubation at 28°C, numbers of sprouts infected with M. terrestris were recorded and percent infection was calculated. RESULTS

Effect of carbon sources on growth of M. terrestris. In a preliminary experiment, pectin or carboxymethyl cellulose

VOL. 56, 1990

SELECTIVE MEDIUM FOR ISOLATION OF M. TERRESTRIS

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FIG. 1. Recovery of M. terrestris from lake sediment soil suspension on dilution plates. Colonies of M. terrestris are dark purple with a gray elevated colony center. Arrows mark all colonies of other soil fungi. (A) Soil dilution on basic rose bengal agar. (B) Soil dilution on MtSA. (C) Infected alfalfa sprouts on MtSA. (D) Vertical rows, different inoculum sources: right, mycelial suspension; middle, agar disk; left, soil suspension. Horizontal rows, different media compositions: top, basic rose bengal agar; middle, MtSA without PCNB and Truban; bottom, MtSA.

or both supported growth of M. terrestris. Hyphal growth was significantly more dense on the Czapek agar containing pectin (to replace sucrose) and rose bengal. However, these carbon sources were not subsequently used in the development of the selective medium because the unique color and appearance of M. terrestris colonies on the medium with rose bengal agar was lost when sucrose was replaced by pectin or carboxymethyl cellulose or both. Effect of single fungicides on growth and recovery of M. terrestris and contaminant fungi from soil dilutions. M. terrestris was generally more sensitive than the other soil fungi to essentially all of the fungicides tested (Table 1). The fungicides Captan, Benlate, tannic acid, gallic acid, and coumarin inhibited M. terrestris almost completely at concentrations which were not effective against the other fungi. Dodine seemed to support growth of both M. terrestris and the undesired soil fungi. Therefore, these fungicides were not subsequently included in the selective medium. Colonies of M. terrestris produced a brown halo in the medium

containing >300 ,ug of cinnamic acid per ml; no other fungi produced a similar halo. This property could be considered beneficial for selective recognition of M. terrestris colonies. In practice, however, when cinnamic acid was added to the complete medium at these concentrations, both M. terrestris and all other fungi were completely inhibited. Recovery of M. terrestnis on MtSA. None of the fungicides tested could completely inhibit the undesired fungi while enabling detectable growth of M. terrestris (Table 1). The medium tested subsequently contained a combination of five fungicides (Table 2). This medium restricted growth of M. terrestris, which aided in its enumeration and identification on the selective medium. The medium also considerably (but not completely) inhibited growth of the undesired fungi. The efficiency of recovery of M. terrestris declined with increasing concentrations of soil. The efficiencies of recovery of M. terrestris from soil dilutions of 2 x 10-3 and 5 x 10-3 was 57 and 39% on MtSA compared with 26 and 9%, respectively, on the less selective basic medium (slightly modified MRBA)

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SNEH AND STACK

APPL. ENVIRON. MICROBIOL.

TABLE 3. Recovery of M. terrestris' and inhibition of other fungi on the selective medium and effect of omitting each fungicide from the medium % of control

Treatmentb

Basic medium SM-Sub SM-Ox SM-Ter SM-T SM-SA SM

M. terrestris

M. terrestris Soil fungi M. terrestris + soil fungi alone, linear CFU/ Linear M. terrestris Soil fungi growth plate growth CFU/plate CFU/plate

100 28 24 60 24 20 20

100 59 90 79 67 82 82

100 29-75 57-100 43-83 29-67 14-33 14-57

TABLE 4. Use of alfalfa sproutsa as baits for isolation of M. terrestris from soil in aquatic conditions, on the selective medium

100 100 142 200 228 371 429

100 76 90 87 78 72 63

a Hyphal fragments (grown in liquid culture) were mixed in soil before dilutions were made. b SM, Selective medium; Sub, Subdue (0.5 ml/liter of 25.1% a.i.); Ox, oxgall (5 g/liter); Ter, Terraclor (0.5 g/liter); T, Truban (0.05 ml/liter of 40.7% a.i.); SA, sorbic acid (35 mg/liter).

(Table 2). The lowest soil propagule concentration of M. terrestris that can be counted on dilution plates of MtSA was around 103/g of soil. Colonies of M. terrestris strongly absorbed the rose bengal, which turned M. terrestris colonies purple with a small gray peak in the center (Fig. 1). On this medium, M. terrestris mycelium was not aerial and could be easily distinguished from other fungal colonies originating from soil. Contaminant colonies were less dense, and most had distinctive aerial mycelia. To determine whether all five fungicides were required in the selective medium, a final experiment was carried out; in each treatment, one of the fungicides was omitted. Results (Table 3) indicate that each fungicide was essential for increasing the recovery of M. terrestris up to 429% of that on basic medium and for considerably reducing the size and recovery of the undesired fungi to 63%. In addition, M. terrestris colony size was restricted by 43 to 86% compared with that on the basic medium. The final composition of MtSA per liter was as follows: KH2PO4, 0.5 g; MgSO4 7H20, 0.5 g; dextrose, 10.0 g; peptone, 5.0 g; chloramphenicol, 0.25 g; rose bengal, 50 mg; oxgall, 5.0 g; Terraclor (PCNB 75% a.i.), 0.5 g; agar, 15.0 g. After autoclave sterilization, the following ingredients were aseptically added: sorbic acid (0.7% autoclave-sterilized aqueous solution), 5.0 ml; Subdue (25.1% emulsion of metalaxyl), 0.5 ml; Truban (40.7% suspension of etridiazol), 0.05 ml. Enumeration of M. terrestris propagules from soil by the drop plate method (4) gave similar results to enumeration by dilution plates on the new selective medium. Recovery of M. terrestris from soil on baits (Fig. 1C). Preliminary experiments indicated that alfalfa sprouts incubated in soil submerged in water were better colonized by M. terrestris than by other fungi. Alfalfa sprouts were better baits than milfoil shoot segments, which were more colonized by other fungi. Also, colonization was higher after 3 days of incubation than after 1 or 2 days. Increasing propagule density of M. terrestris resulted in an increase in colonization percentage of alfalfa sprouts from 5 to 67.5% at concentrations of 1.5 x 102 and 1.5 x 104 propagules per g of soil, respectively (Table 4). The highest colonization percentage of alfalfa sprouts occurred after 3 days of incubation. Colonization by M. terrestris declined after longer incubation periods, while colonization by other fungi in-

concn

Infected alfalfa sprouts (%) Other fungi 5 days 7 days 3 days 5 days 7 days

M. terrestris

soil)'

3 days

1.5 x 104 1.5 x 103 1.5 x 102

67.5

50.3

50.3

1.3

7.3

8.5

11.5

4.0

6.5

1.3

0.0

3.5

5.0

1.3

0.0

0.0

1.3

1.3

15

0.0

0.0

0.0

1.5

0.0

1.5

a Three-day-old alfalfa sprouts were incubated in soil submerged in water at room temperature and 12-h fluorescent photoperiod. b Artificially infested with hyphal fragments.

creased, especially at the highest M. terrestris propagule concentration. DISCUSSION Identification of M. terrestris on the selective medium was based on the intensive absorption of rose bengal by the pathogen's hyphae. The unique purplish colony color of the mature hyphae, the hyaline appearance of the young surrounding hyphae, and the submerged hyphae of M. terrestris on this medium (Fig. 1A) facilitated its identification from the other soil fungi. Attempts to use more selective carbon sources in the selective medium, such as pectin or carboxymethyl cellulose, resulted in loss of the unique color of M. terrestris colonies. This interfered with their positive identification. M. terrestris was generally more sensitive than other soil fungi to all of the fungicides tested. Therefore, some of them such as Captan, Benlate, tannic acid, gallic acid, and coumarin were not suitable for the isolation of M. terrestris. Metalaxyl and etridiazol were used to inhibit pythiaceous fungi, which are active in aquatic environments. The combination of etridiazol, metalaxyl, oxgall, PCNB, and sorbic acid was required in the selective medium to get satisfactory growth of colonies of M. terrestris and inhibit the undesired fungi. When one of these ingredients was omitted from the complete selective medium, the interfering soil fungi grew better and inhibited the colonies of M. terrestris. Although undesired soil fungi still appeared on the new selective medium plates, they were strongly restricted. This restriction allowed more effective recovery of colonies of M. terrestris. The higher the concentration of soil in dilutions, the more other soil fungi interfered with the growth and identification of M. terrestris on the plates. Recovery of 57% of M. terrestris propagules could be obtained from soil dilutions of 2 x 10-3. The drop plate method is essentially an efficient procedure for viable counts of bacteria. The fungal colony restriction properties of the new selective medium enabled the use of this method for counts of propagules of M. terrestris in soil dilutions. This selective medium may be suitable for isolation of M. terrestris from plant or other organic material and from soil when it is present in relatively high concentrations (>103 propagules per g, e.g., in studies on the behavior and survival of M. terrestris under different environmental con-

ditions). To detect lower population levels of this fungus present in soil and in aquatic environments, an attempt was made to

VOL. 56, 1990

SELECTIVE MEDIUM FOR ISOLATION OF M. TERRESTRIS

develop a baiting method. Alfalfa sprouts were therefore evaluated as baits for the recovery of M. terrestris at inoculum densities lower than the recovery limit of soil dilution MtSA plates. However, although the fungus could be recovered from infected alfalfa sprouts, a high inoculum density (104 CFU/g of soil) was required to get an appreciable percentage (67%) colonized. The baiting method could not provide quantitative evaluation of inoculum density but could recover the fungus at lower inoculum densities (1.5 x 102 CFU/g of soil) than by the soil dilution plates on MtSA.

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LITERATURE CITED 1. Gunner, H. B. 1983. Microbiological control of Eurasian watermilfoil. Aquatic Plant Control Research Program Miscellaneous Paper A-83-4. U.S. Army Engineers W.E.S., Vicksburg, Miss. 2. Gunner, H. B., Y. Limpa-Amara, and P. J. Weilerstein. 1985. Microbiological control of Eurasian watermilfoil. Annual Report to Aquatic Plant Control Research Program. U.S. Army Engineers W.E.S., Vicksburg, Miss. 3. Martin, J. P. 1950. Use of acid rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69:215-232. 4. Reed, R. W., and G. R. Reed. 1948. Drop plate method for counting viable bacterial cells. Can. J. Res. Sect. E 26:317-326.