Maritime MicroBiologicals Inc. - Control of Microbial Pathogens

Maritime MicroBiologicals Inc. is developing environmentally-benign ways of controlling pests in agriculture and forestry. Among these, some of our biological control agents show particular promise.

One of these allows mushrooms growers to control "green mould" without the use of fungicides. Its development was funded by the Canadian Mushroom Growers' Association and the National Research Council's IRAP program. (Thank you).

Mushrooms are grown on specially made compost, this providing the complex mix of micro-organisms (both living and dead) and nutrients that favour the growth of the mushroom's mycelium over that of competing micro-organisms. Sometimes however, things get out of balance and mushroom pathogens grow. Green Mould, caused by particular strains of Trichoderma harzianum (Th3 and Th4) is the result of the most serious of these pathogens. T. harzianum attacks the mushroom mycelium and the mushrooms, and can sometimes destroy the entire crop.

In our early work, we confirmed the results of others who had shown that T. harzianum would not grow on mushroom compost unless spawn grains or mushroom mycelium were also present. Interestingly, we found this situation changed markedly if the compost was pre-treated with antibiotics that killed the bacteria in it. In treated compost the green mould completely over-ran the compost within a few days (see photo 1 at right). This suggested that compost bacteria normally keep the green mould under control.

We isolated many compost bacteria and assessed their effect on T. harzianum by growing the two micro-organisms together on agar. Many of them made compounds that diffused into the agar and stopped T. harzianum growth (see plates on left of photo 2). We called these antifungal compound producing bacteria, or ACB's. Two ACB's, which we called isolates 4 and 7 are shown in the photo. We also found that when these bacteria were grown in liquid culture, the culture fluid inhibited the T. harzianum even after considerable dilution.

We have isolated and characterized some of the inhibitory compounds made by these bacteria. The compounds retain activity after autoclaving (i.e. they are relatively heat stable). They are reactive with ninhydrin (indicative of the presence of amino groups), and they co-migrate with certain peptidyl-glycans on thin-layer chromatography plates. We suspect they may be iturins.

Although many other compost isolates were active against the T. harzianum, isolates 4 and 7 were of particular interest since they did not inhibit A. bisporus (the mushroom). When A. bisporus was co-cultured on plates with these bacteria, it grew into the bacterial colony (see petri plates on right of photo). The compounds produced by the bacteria when it was in culture changed the morphology of the A. bisporus mycelium, inducing it to form cords (aggregates of hyphae) and to grow more rapidly.

These results suggested that these isolates (and/or the products made by them) could be used to control green mould of mushrooms. We have tested this idea at the lab scale. Results have been very encouraging. Spawned compost to which the bacteria is added resists growth of an aggressive isolated of T. harzianum (Th 4), even when a large amount of the pathogen is injected into or sprinkled onto the compost.

We are looking for partners who will contribute towards the larger-scale testing that will be required prior to commercialization. Some further background information is available in some of the publications lsisted below.

In another project that was done with support from the New Brunswick Provincial Government (Strategic Partner Development Program), 3M Canada and the National Research Council's IRAP program we did analogous work, but directed towards finding a control for Phytophthora infestans, the cause of potato late blight. The literature shows that when compost is included in plant growing medium, or when plants are sprayed with compost infusion (compost tea), the plants often become more disease resistant. Some reports show that compost tea can control late blight. It is generally recognized that disease control by the compost or the tea results from the activity of bacteria.

We investigated the prevalence of ACB's in a variety of composts, and found they were common. Some were isolated, grown in liquid culture, and the broth was included in various proportions (serial 2x dilution) in agar medium. This was autoclaved, inoculated with test fungal pathogens, and their radial growth rates were measured.

Table 1. Dilution factor giving 50% reduction of the radial growth rate of fungi.
	*ACB Isolate*
*Fungus*	A	B	C	D	E	F
P. infestans	>128	6	8	<16	4	4
T. harzianum	<4	8	8	>128	32	4

In a first set of tests, the various bacteria were compared for their effects on growth of Phytophthora infestans (late blight), and Trichoderma harzianum (mushroom green mould, see above). The results (Table 1) showed that the culture fluid from ACB A was very active at inhibiting growth of P. infestans; it had to be diluted more than 128 fold before the fungus showed half the radial growth rate it showed on agar without added bacterial culture fluid. At lesser dilutions growth was completely eliminated. In contrast, T. harzianum was relatively insensitive to medium from ACB A, only being inhibited if it was included as a large proportion (>1/4) of the medium. However, with ACB D the converse was true. (ACB D is the same as isolate 7 referred to above). Medium from this bacterium inhibited T. harzianum strongly (dilution factor of > 128) while having relatively little effect on P. infestans. The other ACB's had intermediate effects, but culture fluid from all of them was somewhat inhibitory to both fungi.

Table 2. Dilution factor for ACB A and D for ca 50% reduction of the radial growth rate of various fungi.
Fungus	ACB A	ACB D
Pythium ultimum	>128	8
Fusarium sambucinum	4	4
Botrytis cinerea	32	64
Alternaria solani	32	64

We extended these findings by testing the medium from liquid cultures of ACB’s A and D against some other pathogens. The results (Table 2) showed that both had some inhibitory effect against all the fungi, but that A was particularly active against P. ultimum, while D was most active against B. cinerea and A. solani.

We are continuing to test ACB's and preparations made from them for their abilities to control pathogens on plants. A key to success is formulation. Preparations from ACB A have shown fairly good activity against late blight. For example, in one test using excised potato leaves, when leaves were sprayed with water (positive control) about 90% of them became covered with sporangia 5 days after inoculation with P. infestans. In leaves sprayed with the preparation from A, only about 10% of the leaves showed signs of being infected. As mentioned above, ACB D shows good potential to control the mushroom disease Green Mould, caused by T. harzianum.

In a complimentary part of the project we tested various natural plant products for their ability to control P. infestans (late blight) on stored potato tubers or on potato plants. Here our focus was on natural products, including various monoterpenes, sesquiterpenes and essential oils that are generally recognized as being safe (EPA GRAS status). Since some of the compounds (particularly the monoterpenes) were too volatile to be of practical use, controlled-release formulations (proprietary technology) were provided by 3M-Canada.

Results with some of these preparations were encouraging. For example, some of the formulated monoterpenes completely stopped growth of P. infestans on stored potato tubers for at least four months, even when the tubers were repeatedly inoculated with P. infestans sporangia. The same preparation also completely stopped the tubers from sprouting. When similar preparations were sprayed onto leaves, they conferred blight protection for about 10 days, without any obvious effect on the plants' ability to grow or produce potatoes.

We are looking for financial support and technical expertise that will allow us to continue developing these products. If you are interested in additional information about our blight control technology, please contact us.

Some of our publications and reports concerning pathogen control.

Boyle, D. 1999.Why mushrooms are not wiped out by green mould. Mushroom World.10(4):5-10.

Boyle, C.D. 1998. Testing essential oils and other chemicals for use against green mould. Mushroom World 9:51-57.

Boyle, D. and G. Lonergan. 2001. Controlling Phytophthora infestans and other field and storage pathogens of potato using novel formulations of essential oils. Final Report. TDF program. NBDARD and 3M Canada (Copy available on request).