ABSTRACT

Paenibacillus polymyxa (syn. Bacillus polymyxa) strain PKB1 inhibited Leptosphaeria maculans, the causal fungus of blackleg of canola (Brassica napus and B. rapa). PKB1 was found to produce two closely related antifungal peptides with eight amino acids and masses of 835.5 and 897.5 Da. In vitro, PKB1 significantly reduced germination and germ-tube length of L. maculans pycnidiospores. PKB1 also inhibited several other fungi pathogenic on canola, including Alternaria brassicae, Fusarium avenaceum, Pythium sp., Rhizoctonia solani AG2-1, and Sclerotinia sclerotiorum. PKB1 significantly suppressed blackleg disease development on B. napus cv. Westar stems when the leaves were inoculated with a suspension of PKB1 cells and L. maculans pycnidiospores. In field experiments, PKB1 and propiconazole, individually and in combination, significantly reduced formation of pseudothecia and ascospores of L. maculans, and decreased survival of the fungus on infected canola stubble. Several fungicides (propiconazole, prochloraz, iprodione) and herbicides (clopyralid, ethametsulfuron-methyl, sethoxydim) registered for use on canola were found non-deleterious to growth of PKB1 in vitro. PKB1-amended compost inhibited germination of Sclerotinia sclerotiorum sclerotia in growth chamber experiments, and reduced viability and pseudothecia formation of L. maculans under field conditions. Eight PKB1-specfic primers constructed using the RAPD-PCR technique were developed to allow quantification of the bacterium in compost. PKB1-amended compost demonstrated good potential as a biocontrol agent against several soil-borne diseases of canola.

INTRODUCTION:

Blackleg caused by the highly virulent strain of Leptosphaeria maculans (conidial state: Phoma lingam) is the most important disease of canola (Brassica napus and B. rapa) in Canada and several other temperate countries. The disease is seed-borne and survives on infected canola stubble. Fungicidal seed treatments and foliar applications of fungicides such as propiconazole do not completely control the disease (Kharbanda 1992).

Very little has been published regarding biological control of blackleg of canola/rapeseed. Partial suppression of virulent L. maculans with the weakly virulent strain of the pathogen in vitro and in vivo has been reported (Petrie 1982). A strain of Penicillium verrucosum was found to produce a metabolite toxic to L. maculans (Kharbanda and Dahiya 1990). Recently, we recovered a strain of Paenibacillus polymyxa (PKB1, syn. Bacillus polymyxa) from canola roots, which was found highly inhibitory to the growth of L. maculans in vitro. This research was undertaken to explore the use of this bacterium, alone or in combination with fungicides or in compost, to control blackleg and other diseases of canola. Brief reports of this have been presented (Yang et al. 1998, Kharbanda et al. 1997).

OBJECTIVES:

(1) To characterize the antifungal agent, and determine conditions for its optimal production.

(2) To determine if use of the bacterium alone or in combination with fungicides or in compost can control blackleg and other diseases of canola.

(3) To develop a molecular probe for detecting the bacterium in amended compost and soil.

CHARACTERIZATION OF THE ANTIFUNGAL AGENT:

The antifungal agent was found to be an N-terminally blocked peptide of approximately 2000 Da. The agent is soluble in methanol and acetic acid but not in water, suggesting that it may have a fatty acid component. However, it is active in water; sporulating colonies can inhibit the growth of L. maculans on aqueous based agar medium. It is stable at temperatures ranging from -70°C to 100°C and is made up of eight amino acids. Two peptide components, one with a mass of 883.5 Da and the other with a mass of 897.5 Da were identified. The peptide is most likely a cyclic compound.

EFFECT OF THE BACTERIUM ON GROWTH OF L. MACULANS:

L. maculans pycnidiospore germination and germ tube length were significantly reduced by PKB1 when tested on autoclaved cellophane membranes (Table 1).

Table 1. Effect of Paenibacillus polymyxa PKB1 on pycnidiospore germination and germ tube length of Leptosphaeria maculans (BLA).

	Percent of pycnidiospore germination*		Percent of pycnidiospore germination**
Day	BLA+PKB1	BLA	BLA+ PKB1	BLA
1	12.25 a	79.50 b	3.905 a	11.835 b
2	18.25 a	96.50 b	8.595 a	15.165 b
3	23.50 a	98.50 b	9.625 a	83.665 b

* Based on 400 pycnidiospores.

** Average of 100 spores. Means within individual rows followed by the same letter are not significantly different determined by a Duncan’s Multiple Range Test (P=0.05).

In growth chamber tests, two-week old seedlings of B. napus cvs. Quantum and Westar were inoculated with suspensions of pycnidiospores or PKB1 cells mixed with pycnidiospores. Disease severity was assessed after incubation for six weeks at 20°C. PKB1 reduced blackleg incidence and severity on the blackleg susceptible cultivar Westar but not on the resistant cultivar Quantum (Table 2).

Table 2. Effect of PKB1 on blackleg disease development caused by Leptosphaeria maculans (BLA) on canola plants in a growth chamber test.

Cultivar	Treatment	Disease incidence	Disease severity
Westar	Sprayed with BLA pycnidiospore suspension	100.0 a*	3.64 a
Westar	Sprayed with BLA and bacterial suspension	56.9 b	1.28 b
Quantum	Sprayed with BLA pycnidiospore suspension	8.3 c	0.08 c
Quantum	Sprayed with BLA and bacterial suspension	5.8 c	0.06 c

* Means in columns followed by the same letter are not significantly different according to a Duncan’s Multiple Range Test (P=0.05).

EFFECT OF PROPICONAZOLE AND PKB1 ON SURVIVAL OF L. MACULANS ON CANOLA STUBBLE

Infected canola stubble in pots was sprayed with either propiconazole (125 g a.i./ha) or PKB1 suspension (7.4 × 10⁷ cells/ml) and incubated at temperatures ranging from -5° to 20°C. Stubble in half the pots was covered with sterilized soil mix to simulate stubble buried outdoors. Blackleg disease severity and viability of L. maculans pycnidia was determined at the end of the incubation period on each stubble piece. Propiconazole significantly reduced the number of pycnidia under all temperature regimes (except 20 °C, unburied), but the bacterium had minimal effect. However, PKB1 significantly reduced L. maculans survival under most conditions in comparison to untreated or propiconazole treated stubble.

EFFECT OF PKB1 ON OTHER FUNGAL PATHOGENS OF CANOLA:

Effectiveness of the Paenibacillus sp. strain was tested against Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria brassicae, Pythium sp., and Fusarium sp. by measuring fungal inhibition zones on Potato dextrose (PD) agar plates and by determining mycelium dry weight in PD broth.

In Petri plate and liquid culture tests, the bacterium Paenibacillus sp. was found to have significant inhibitory effects on all fungi tested (Table 3).

Table 3. Effect of PKB1 on mycelial growth of some fungal pathogens of canola.

		Mean mycelial dry weight (mg)
Isolate	Pathogen	With bacterium	Control
S94-1	Sclerotinia sclerotiorum	5 a*	152 b
S94-2	S. sclerotiorum	3 a	89 b
88-1-8	Pythium pythioides	16 a	94 b
C51-25	Rhizoctonia solani AG2-1	13 a	147 b
R21	R. solani AG2-1	9 a	153 b
N15-6	Fusarium avenaceum	4 a	53 b
P66-30	F. avenaceum	3 a	43 b
Alt-1	Alternaria brassicae	3 a	80 b
CA2	A. brassicae	4 a	21 b

* Mean of four replications; values within a row followed by the same letter are not significantly different determined by Tukey’s Studentized Range Test (P=0.05).

EFFECT OF FUNGICIDES AND HERBICIDES ON PKB1:

This was determined by placing fungicide-treated filter paper discs on (PD) agar plates pre-seeded with the bacterium and measuring inhibition zones after one week incubation at 23°C.

All herbicides (clopyralid, ethametsulfuron-methyl, sethoxydim) and fungicides (propiconazole, prochloraz, iprodione) tested were found to have no inhibitory effect on the bacterium.

EFFECT OF PKB1-AMMENDED COMPOST AND PROPICONAZOLE ON LEPTOSPHAERIA MACULANS:

Near mature composts (cattle manure and wood chips) were pasteurized by steaming 0.5 m³ of each for 5 hours at 80 °C in a soil steamer, then inoculated with a suspension of freeze-dried PKB1 spores (1 × 10⁹ /ml). The amended composts were then incubated for 45 days to enhance the population of PKB1.

The effect of various combinations of PKB1, propiconazole and compost on viability of stubble-borne L. maculans was determined by placing treated stubble outdoors and retrieving samples every six months. The viability of the fungus was found to have been significantly reduced in stubble treated with propiconazole, propiconazole + PKB1, and PKB1 + compost. There were significant differences in pseudothecia production in response to treatments and burial methods in samples retrieved after 18 months. Compost + PKB1 and propiconazole + PKB1 had a significant inhibitory effect on ascospores formation on canola stubble.

EFFECT OF PKB1-AMMENDED COMPOST AND PROPICONAZOLE ON SCLEROTIA OF SCLEROTINIA SCLEROTIORUM:

When sclerotia were buried in compost amended with PKB1, their germination was significantly inhibited by the presence of PKB1. Certain batches of compost gave better results than others and were equally effective without the presence of PKB1.

DEVELOPMENT OF A MOLECULAR PROBE:

DNA of PKB1 and other 22 isolates from eight different Paenibacillus species was amplified with random primers. Four DNA fragments were selected to make DNA probes. Specificity of the probes was detected by Southern blotting. Four DNA probes were cloned and sequenced. Primers were designed according to known oligonucleotide sequences and tested on the 23 bacterial isolates. Eight primer pair combinations gave similar results and amplified only one DNA band (approximately 500 bp) of PKB1 and strains of other Paenibacillus species (Figure 1).

Figure 1. PCR products of 23 strains from eight Paenibacillus spp. amplified with specific primers.

CONCLUSIONS:

• The antifungal agent produced by the bacterium PKB1 is most likely is a cyclic peptide and is heat resistant.

• PKB1 is capable of inhibiting growth of different isolates of several fungi that cause important diseases on canola such as blackleg, Rhizoctonia root rot, Sclerotinia stem rot and Alternaria black spot.

• Most chemicals used on canola do not have deleterious effects on growth of PKB1.

• Propiconazole significantly reduced the number of pycnidia on stubble and the bacterium significantly reduced survival of L. maculans under growth chamber conditions.

• The biocontrol agent could be differentiated from other P. polymyxa strains using RAPD-PCR.

• Compost could be a useful carrier to deliver PKB1, however it needs further developmental work.

ACKNOWLEDGEMENTS:

This project was partly supported by the Alberta Canola Producers Commission and the Alberta Agricultural Research Institute. Technical assistance from Matthew Ostashewski and Sam Werezuk is gratefully acknowledged. We thank Ralph Lange for reviewing the manuscript.

REFERENCES:

1. Kharbanda, P.D. 1992. Performance of fungicides to control blackleg of canola. Canadian Journal of Plant Pathology 14:169-176.

2. Kharbanda, P.D. and J.S. Dahiya. 1990. A metabolite of Penicillium verrucosum inhibitory to growth of Leptosphaeria maculans and Rhizoctonia solani. Canadian Journal of Plant Pathology 12:335 (Abstract).

3. Kharbanda, P. D., J. Yang, P. H. Beatty, S. E. Jensen, and J. P. Tewari. 1997. Potential of a Bacillus sp. to control blackleg and other diseases of canola. Phytopathology 87 (6): S51 (Abstract).

4. Petrie, G.A. 1982. Blackleg of rapeseed (canola) caused by Leptosphaeria maculans: interaction of virulent and weakly virulent strains and implications for biological control. Canadian Journal of Plant Pathology 4:309 (Abstract).

5. Yang, J., P. D. Kharbanda, and J. P. Tewari. 1998. Development of specific primers to a biocontrol agent against Leptosphaeria maculans. Presentation at the annual meeting of the American Phytopathological Society, Las Vegas, NV. U.S.A. Nov. 8-12, 1998.