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World Journal of Microbiology & Biotechnology 18: 661–671, 2002.  2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Microbial studies of compost: bacterial identification, and their potential for turfgrass pathogen suppression Jeanine I. Boulter , Jack T. Trevors* and Greg J. Boland  Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 *Author for correspondence: Tel.: þ1-519-824-4120, Fax: þ1-519-837-0442, E-mail: [email protected]   Also corresponding authors Received 12 December 2001; accepted 17 April 2002

Keywords: Bacteria, compost, biocontrol disease suppression, grey snow mould, diversity, fungi pathogen, pink snow mould, phospholipid analysis, plant microorganism, turfgrass

Summary Composting is the degradation of organic materials through the activities of diverse microorganisms. This research examined microbial community dynamics, population levels and identification of bacteria throughout the composting process and in storage. In addition, an evaluation was performed to determine the potential for dominant bacterial isolates to suppress selected turfgrass pathogens: Sclerotinia homoeocarpa, Pythium graminicola, Typhula ishikariensis, and Microdochium nivale, responsible for causing the turfgrass diseases dollar spot, pythium blight, typhula blight, and fusarium patch, respectively. Composts supported high population levels of bacteria with 78% of cultures tested being Gram-negative. Proteolytic activity, found in 29% of cultures tested is a potential mechanism of suppression or competition with other microorganisms. Although the Biolog system did not identify a wide range of bacteria, the main Gram-negative genera identified in mature compost were Pseudomonas (28%), Serratia (20%), Klebsiella (11%), and Enterobacter (5%). Twenty-one percent of isolates tested were not identified by Biolog, and many more had similarity indexes log 9 (n=5, except for one treatment). a DAH refers to time after harvest of mature composts. b n refers to the number of plates that were read for the results listed. c NA – nutrient agar media. d PDA – potato dextrose agar.

NA and 8:38  0:31 log c.f.u./g on PDA after 48 h. At 36 days after harvest (DAH) of mature compost, counts were equal to or greater than 8.85 on NA and 7:28  0:28 on PDA after 48 h. At 89 DAH, counts were equal to or greater than 8:89  0:12 on NA and 8:11  0:27 on PDA after 48 h (Table 2). Microbial counts remained high during extended storage of composts. A culture collection was created by selecting prevalent colonies and subculturing. Results of the Gram-reaction, proteolytic test, and Biolog procedure are presented in Table 3. Of the bacterial cultures tested for their Gram-reaction, 83% were Gram-negative. Twenty-nine percent of cultures tested for gelatin hydrolysis/proteolytic activity were positive (includes Gram-positive strains not included in Table 3, since they were not tested for identification with Biolog). Biolog testing, based on biochemical test results, was performed on the Gram-negative bacterial cultures. The results include the genus and species of all cultures tested, although the similarity index was not always high enough to identify to the species level. Results with a similarity index >0.50 are considered positive identifications, while those with a similarity index 80% of total PLFAs) being terminally branched saturated (Ter Br Sats) fatty acids, representative of mainly Gram-positive bacteria. These levels decreased as compost internal temperatures declined, and further declined during storage. Monoenoic (Monos) PLFAs, characteristic of Gram-negative bacteria, were low while composting temperatures were high (day 200) and increased steadily as compost approached harvest (day 306), and during storage. Mid-chain branched saturated (Mid Br Sats) PLFAs, common in Actinomycetes spp., sulphate-reducing bacteria, and certain Gram-positives also increased steadily, to >16% of total PLFAs, as composting compost approached harvest and during storage. Results of plate challenge experimentation on S. homoeocarpa, P. graminicola, M. nivale and T. ishikariensis are presented in Table 5. Of 94 bacterial isolates tested, 20% produced inhibition zones of >10 mm between individual bacterial colonies and that of S. homoeocarpa, which included (in order from greatest to least distance) cultures 65, 99, 55, 49, 59.1, 50, 43.1, 43.2, 97.2, 77, 98, 93, 37, 28, 44, 16 and 78.1. Eleven percent of the isolates produced inhibition zones of >10 mm distance between individual bacterial colonies and that of P. graminicola, which included (in order from greatest to least distance) 43.1, 43.2, 93, 44, 99, 28, 48.1, 55, 59.1, and 49. Eleven percent of the isolates produced inhibi-

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Microbial study of compost Table 3. Results of Gram-reaction, proteolytic test, and Biolog procedure (Biolog Inc., Hayward California) testing for bacteria cultures. Culture numbera

Gram-reaction

Gelatin hydrolysis

Identity (Biolog)

Similarity to Biolog reference strain

5 7 12 14 16.1 19 26 28 31 36 37 41 43.1 45 46 48.1 50 51 53.1 53.2 53.3 55 60 63 64 67 71 72 73 75 77 78.1 78.2 79 80 82 85 86 87.1 90 91.1 91.2 92 94.1 94.2 97.2 103.2 105 110 111.1 114 116 118 122 123 124 125 133 134 135.2 152

                                                            

  + + + +  + ±c   + + + + + +     +    +                     + +     ± +  ± ± ±   ±

Klebsiella pneumoniae ss. pneumoniae Enterobacter aerogenes Serratia marcescens S. marcescens Pseudomonas synxantha P. marginalis Achromobacter cholinophagum P. aeruginosa S. marcescens Stenotrophomonas maltophila P. fluorescens biotype C S. marcescens P. fuscovaginae E. gergoviae S. marcescens P. aeruginosa S. marcescens S. rubidaea Aeromonas veronii S. marcescens A. veronii S. odorifera A. cholinophagum S. marcescens Xanthomonas oryzae pv oryzae P. fluorescens biotype G P. mendocina K. pneumoniae P. fluorescens Salmonella subspecies 1E S. marcescens P. synxantha P. fluorescens biotype G P. chloroaphis S. ficaria S. rubidaea K. pneumoniae S. marcescens P. fulva S. ficaria S. marcescens P. fragi K. pneumoniae P. synxantha P. synxantha P. synxantha K. pneumoniae ss. ozaenae K. pneumoniae ss. pneumoniae Acinetobacter calaoaceticus P. fluorescens A. cholinophagum P. fluorescens B E. gergoviae K. pneumoniae K. pneumoniae K. pneumoniae P. corrugata P. putida biotype B Kluyvera cryocrescens P. mendocina E. agglomerans

0.62 0.54 0.71 0.71 0.71 0.67 0.49 0.77 0.57 0.52 0.84 0.64 0.46 0.89 0.67 0.65 0.55 0.49 0.37 0.38 0.31 0.48 0.74 0.39 0.62 0.84 0.52 0.51 0.33 0.52 0.50 0.34 0.42 0.37 0.51 0.47 0.45 0.22 0.83 0.86 0.47 0.30 0.38 0.07 0.61 0.70 0.55 0.54 0.49 0.81 0.61 0.81 0.20 0.54 0.50 0.30 0.76 0.25 0.18 0.63 0.32

Biolog identity is listed along with the similarity to reference strain. For the Biolog similarity index, values >0.50 are considered positive identifications, and those with a similarity index 10 mm between individual bacterial colonies and that of M. nivale, which included (in order from greatest to least distance) 49, 99, 59.1, 65, 47.2, 91.1, 93, 43.1, 98, and 43.2. Eleven percent of the isolates produced inhibition zones of >10 mm between individual bacterial colonies and that of T. ishikariensis, which included (in order from greatest to least distance) 65, 79, 49, 93, 43.1, 48.1, 98, 43.2, 50, and 44. There were 48, 69, 68, and 81% of the bacterial isolates with no antagonistic activity against S. homoeocarpa, P. graminicola, M. nivale, and T. ishikariensis, respectively (i.e. the distance between the pathogen and isolate was 0 mm) (data not shown). Discussion In this study, microbial counts remained high throughout the storage of composts. High microbial activity in

Figure 1. Diversity of microbial communities in composts 6 and 9 as estimated by PLFA analysis. Days 200, 230 and 306 refer to days after start of composting, with day 306 being the day of harvest, when compost piles were screened and bagged for storage. Day 561 refers to days into storage of compost, after harvest. Terminally branched saturated (Ter Br Sats) are PLFAs representative of Gram-positive bacteria, but may also occur in the cell membranes of some Gramnegative bacteria. Monoenoics (Monos) are PLFAs found in Gramnegative bacteria. Mid-chain branched saturated (Mid Br Sats) fatty acids are common in Actinomycete spp., sulphate reducing bacteria and certain Gram-positive bacteria.

composts is considered to be crucial in suppressive media, hence the enumeration of microorganisms in this research (Hoitink & Fahy 1986; Beffa et al. 1996b). However, possible sources of error in enumeration should be noted: potential overestimation of mycelialforming organisms due to breakup of fragments, selectivity of media, failure of colonies to develop, and possible interactions among organisms (i.e. antibiotic and other antagonistic extracellular chemicals and mechanisms) (Parkinson et al. 1971; Wollum 1982; Chung & Neethling 1988; Tunlid et al. 1989). Despite these difficulties, plate counts are useful techniques for isolating bacteria for evaluation as potential antagonists to pathogens and determining mechanisms of action. No microbiological technique is as widely accepted in enumerating soil organisms as the plate counting method using a variety of media (Wollum 1982). The majority of bacteria isolated from composts were Gram-negative. Other research has also found Gramnegative bacteria and actinomycetes to be more preva-

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Microbial study of compost Table 5. Plate challenge experiment between selected bacteria isolates from compost and S. homoeocarpa, P. graminicola, M. nivale, and T. ishikariensis. Culture numbera

5 7 12 14 16 19 21 28 36 37 40 41 43.1 43.2 44 45 46 47.2 48.1 49 50 51 52.1 52.2 55 59.1 63 65 66 71 77 78.1 78.3 79 86 87.2 91.1 91.2 91.3 93 94.1 97.1 97.2 97.3 98 99 100 103 110 116 123.1 135.2 144 151.1 151.2 LSD (0.05)c

Responseb (mm) S. homoeocarpa

P. graminicola

M. nivale

T. ishikariensis

0.75 0.00 1.75 4.50 10.25 2.25 3.35 10.50 1.00 10.65 6.00 2.70 15.25 14.35 10.46 2.50 1.50 0.00 14.40 16.75 16.20 0.00 7.65 5.40 17.50 16.25 1.25 19.05 3.75 5.75 12.00 10.20 6.15 7.00 6.35 1.10 7.40 0.00 10.30 10.79 1.00 0.60 13.75 9.10 11.95 17.75 3.75 4.50 1.60 0.00 2.25 3.00 0.00 0.85 0.85 1.48

1.00 1.00 2.50 5.45 3.75 0.00 0.00 11.80 0.00 0.00 1.00 2.60 19.70 18.35 12.15 0.00 0.55 0.00 11.75 10.05 9.75 0.00 0.00 0.00 11.00 10.08 0.00 13.00 0.00 0.00 0.00 0.00 0.00 0.00 2.15 0.00 0.00 4.25 0.00 12.95 0.00 0.00 0.00 0.00 8.50 11.90 0.00 0.00 0.00 5.75 0.00 0.00 0.00 0.00 0.00 0.56

7.00 0.00 2.15 8.00 4.75 0.00 0.00 9.45 0.00 0.00 0.00 5.05 12.63 10.40 9.55 3.50 9.10 17.25 25.00 24.25 9.00 0.00 0.00 0.00 8.10 19.55 0.00 18.25 0.00 4.50 0.00 0.00 0.00 0.00 0.00 0.00 15.25 0.00 4.75 14.20 0.00 0.00 0.00 0.00 11.30 19.60 0.00 0.00 0.00 2.25 0.00 2.50 5.65 0.00 0.00 0.40

0.00 0.00 1.60 6.25 0.00 0.00 0.00 7.70 0.00 0.00 0.00 3.75 14.40 12.35 10.25 0.00 1.50 0.00 12.75 16.10 11.25 0.00 0.00 0.00 7.25 6.25 0.00 19.05 0.00 0.00 0.00 0.00 0.00 17.75 0.00 0.00 0.00 0.00 5.50 15.46 0.00 0.00 0.00 0.00 12.55 2.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.16

The response was measured as the average distance, in mm, from streaked bacterial culture to fungal pathogen, on PDA plates, after 4 d for S. homoeocarpa and P. graminicola, 13 d for M. nivale, and 35 d for T. ishikariensis.

lent in suppressive composts compared to conducive media (Tunlid et al. 1989; Atkinson et al. 1997). Kwok et al. (1987) found Gram-negative bacteria to be the predominant control agents in suppressive bark compost. As 29% of the 86 isolates tested were positive for gelatin hydrolysis, it is possible that this proteolytic ability is a mechanism of suppression or competition with other organisms, including turfgrass pathogens. In this study, a number of bacterial cultures became nonviable in storage and were not used in further testing. In addition, a number of viable cultures would not grow on TSA, making use of the Biolog and MIDI difficult. When environmental samples are plated onto TSA, it is not uncommon to observe colonies that cannot be transferred to fresh TSA. This inability to transfer may be due to growth factor requirements or to physical conditions not available in the medium (e.g. proximity to other cells) (Atkinson et al. 1997). Atkinson et al. (1997) found that between 51 and 100% of total colonies from initial spread plate counts could be recultured onto TSA agar. The culturing of predominant bacterial populations was done to identify common species in compost that may be important in suppression of turfgrass pathogens. In this study, Biolog did not identify a wide range of bacteria and had a low percent similarity to the reference strain for many isolates. The main genera identified included Pseudomonas, Serratia, Klebsiella, and Enterobacter. Twenty-one percent of the cultures tested were not identified and others had a similarity index of