Does anaerobic compost contribute to the growth and development of Trichoderma Green Mold?

Observations made over the years at farms in Pennsylvania and elsewhere have suggested that certain mushroom composting (Phase I and/or Phase II) characteristics were often associated with Trichoderma Green Mold disease development. The occurrence of Trichoderma aggressivum biotype Ta2 and Ta4 (T. harzianum, Th2 and Th4), the causal organism of Green Mold, at farms even with a high level of sanitation, appears to be related to compost not being nutritionally selective for the mushroom mycelium. Wet compost, which is poorly aerated during Phase I and Phase II, was often related to increased severity or incidence of disease on the farms.

Both Grogan (1995) and our lab have reported that compost prepared under low oxygen conditions was more susceptible to disease development. Under these low oxygen or anaerobic conditions, bacteria produce organic acids, which may be persistent during the substrate preparation process and residual at the completion of the substrate preparation process when the mycelium of A. bisporus is seeded into the substrate. It has never been reported how organic acids influence the growth of T. aggressivum or whether elevated levels of these acids would influence disease incidence and or severity. We have recently completed a series of experiments to determine the influence of several organic acids on the growth and development of T. aggressivum in culture and in mushroom substrate.

MATERIAL & METHODS

In the first experiment, five organic acids were screened; Lactic ([C.sub.3][H.sub.6][O.sub.3]), Formic (C[H.sub.2][O.sub.2]), Fumaric ([C.sub.4][H.sub.4][O.sub.4]), Succinic ([C.sub.4][H.sub.6][O.sub.4]), and Butyric ([C.sub.4][H.sub.8][O.sub.2] Na) acids at 1 percent, 5 percent and 10 percent in Potato Dextrose Yeast Agar (PDYA). For the second experiment three organic acids were evaluated; Lactic ([C.sub.3][H.sub.6][O.sub.3]), Fumaric ([C.sub.4][H.sub.4][O.sub.4]), and Succinic ([C.sub.4][H.sub.6][O.sub.4]), acids at 0.01 percent, 0.05 percent, 0.10 percent 0.50 percent, 1.0 percent and 5.0 in Compost Agar (CA). For both experiments a measured amount of agar was placed in a 250 ml PDYA and each acid was added at the percent indicated and then autoclaved. A plug from a fresh T. aggressivum culture was placed in the center of each plate. The plates were then placed in an incubator. During incubation at 25[degrees]C, colony radii were measured daily. The examinations were carried out with three replicates for each treatment and the standard deviation (s.d.) values were determined.

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The compost experiments were prepared with Mushroom Test Demonstration Facility (MTDF) compost with Phase I taking place in an environmentally controlled aerated bunker with our standard composting formula consisting of switchgrass hay, straw bedded horse manure, supplemented with poultry manure, distiller's grain and gypsum. During the Phase I aerated composting, the compost is turned on day 3 and filled on day 7 into the Phase II closed tunnel with computer controlled environmental conditions for oxygen, air movement and temperature. In six days when conditioning was completed, the compost was cooled and taken to the Mushroom Research Center (MRC) for spawning.

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At spawning, the Phase II compost was observed to be of medium length, chocolate brown in color, with no detectable odor of ammonia. Compost moisture was judged to be good, and subsequent analysis showed a moisture level of 72.5 percent. Three organic acids were evaluated in this compost; Fumaric ([C.sub.4][H.sub.4][O.sub.4]), Lactic ([C.sub.3][H.sub.6][O.sub.3]) and Succinic ([C.sub.4][H.sub.6][O.sub.4]) acids at 0.005, 0.025 and 0.05 percent based on dry weight. The compost was spawned with a commercial off-white hybrid. At spawning, Phase II compost was weighed (454g), mixed with 1 tsp of spawn and the pre-weighed organic acid. Each tub was labeled and covered with a plastic bag and was secured by a rubber band. The tubs were then placed in an incubator at 25[degrees]C. Two days after the spawning each tub was inoculated with 1 ml of a [10.sup.-5] spore suspension of Trichoderma. Each tub was evaluated daily for presence of T. aggressivum growth. Growth was rated and recorded based on coverage area of the surface of each tub. Growth data was statistically analyzed using the Waller Duncan k-ratio t-test at a significance level of 0.05 to separate the means.

RESULTS & DISCUSSION

The results of the in vitro assays suggested that concentrations above 1 percent of the organic acids tested in PDYA slowed the linear growth of T. aggressivum, Figure 1. The 5 and 10 percent concentrations of Lactic, Succinic and Fumaric acids delayed and slowed the T. aggressivum growth more than did the lower concentrations of those acids. All acids, but Fumaric, at 10 percent concentrations completely inhibited the growth of T. aggressivum. In the second experiment lower concentrations of these acids were used in compost agar, Figure 2. Concentrations of Succinic, Fumaric and Lactic acids at or below 0.1 percent showed an increase in the linear growth rate of T. aggressivum. Fumaric acid seemed to increase the linear growth more than Succinic and Lactic acid, but not significantly different at similar concentrations. Whereas, concentrations of most all acids above 0.5 percent showed an inhibition of growth over a four day period, when compared to the untreated control. Except for Succinic at 0.5 percent and 1.0 percent, the higher concentrations of Lactic and Fumaric acids had a significantly more influence in delaying the growth.

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Compost experiments, Figures 3 and Figure 4, suggested that when T. aggressivum was inoculated into the compost at spawning time, it colonized the compost faster at concentrations at or above 0.025 percent for Lactic, Succinic and Fumaric acids, when compared to the inoculated, untreated compost. In six days the inoculated untreated compost showed no growth of T. aggressivum. Results of the compost bioassays suggested that 0.05 percent concentrations of all acids significantly improved T. aggressivum growth in the substrate when compared to the 0.025 percent and 0.005 percent concentrations. In the second bioassay, Figure 4, the 0.025 percent Succinic acid resulted in a growth rate similar to the 0.05 percent concentrations. In the lower concentration 0.025 percent of all acids also resulted in significantly faster growth than the 0.005 percent concentrations of the acids. Higher concentrations of Fumaric in substrate visually resulted in a more complete and vigorous colonization by T. aggressivum, Figure 5. It was surprising how quickly the Trichoderma grew in six days of the test, whereas the earliest it is usually seen on a commercial farm may be as soon as 10-12 days. In the compost treated with these acids at all concentrations, in six days the spawn grew as expected.

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SUMMARY

Wet, dense compost has been associated with increased incidence of Trichoderma Green Mold disease. During the Phase I composting process, under anaerobic conditions, organic acids are produced by anaerobic bacteria and remain as residual compounds in the compost for the duration of the composting and possibly spawn growing periods. We know that spore load is probably the biggest component of Trichoderma Green Mold disease development. With excellent sanitation growers can reduce or eliminate the incidence of this disease in even marginal compost. With an average sanitation program, compost can become severely infected in both marginal and optimal compost. The results of these culture and compost bioassays suggest that small amounts of organic acids added to a media or compost at spawning will increase the growth rate of T. aggressivum. In compost being colonized by mushroom mycelium, most all concentrations of the organic acids tested resulted in a faster colonization and more severe infestation by T. aggressivum. These results support the theory that anaerobic conditions are a predisposing factor to the incidence and severity of Trichoderma Green Mold disease. Recent comments from a composter/grower who reduced anaerobic conditions on his wharf also stated that it helped to reduce the incidence of Green Mold, supporting this research. Therefore, under commercial conditions compost with poor oxygen conditions in Phase I and possibly in Phase II would become more susceptible to Trichoderma Green Mold.

REFERENCES

Beyer, D. M. 1996. Observations and practices on Pennsylvania mushroom farms that influence the incidence of Trichoderma harzianum. Mushroom Green Mold Round Table. The Pennsylvania State University. Misc. Mimeo. p. 11

Beyer, D.M., M. G. Anderson, N. G. Catlin, J. Kremser and P. J. Wuest. 1998. Identification and evaluation of predisposing factors affecting the occurrence and development of Trichoderma green mold of Agaricus bisporus. International Congress Plant Pathology, Edinburgh, Scotland. Vol. 3 6.4

Fletcher, J.T. 1997. Mushroom spawn and the development of Trichoderma harzianum compost mold. Mushroom News 45 (8) 6-11.

Grogan, H.M., and Gaze, R.H. 1995. Growth of Trichoderma harzianum in traditional and experimental composts. Mush. Science. XIV (2) 653-660.

Rinker, D.L. and Alm, G. 1997. Investigations of factors influencing the expression of green mold. Mushroom World 8 (2): 25-29.

Seaby, D.A. 1996. Investigation of the epidemiology of green mould of mushroom (Agaricus bisporus) compost caused by Trichoderma harzianum. Plant Pathology 45:913-923.

ACKNOWLEDGEMENTS