Biocontrol Potential of Fungal and Oomycete Phytopathogens by Myxobacterial Strains

Abstract

Myxobacteria, a group of swarming, predatory soil bacteria, are of interest because of their biocontrol potential. In this study, the inhibitory effects of 13 strains of myxobacteria were examined against four different phytopathogenic fungi, as follows: two isolates of Rhizoctonia solani from different AG groups and one isolate each from Sclerotinia sclerotiorum and the oomycete Pythium ultimum. Inhibition levels varied among phytopathogens, with slow-growers being more susceptible than fast-growers. Myxococcus xanthus BS 248, M. flavus ATCC 29617, and M. coralloides BS249 were the most inhibitory strains tested. non-contact and contact inhibition on agar media between phytopathogens and myxobacteria were visually discernible. This distinction potentially reflects the activity of low-molecular-weight metabolites and high-molecular-weight lytic enzymes, respectively. In a pot soil study, the inhibitory effect of a mixture of two strains of myxobacteria against two strains of R. solani was apparent from the reduced disease in cucumber seedlings compared to controls without myxobacteria. This is the first report of an in vivo inhibitory effect of myxobacteria against Rhizoctonia. The survival of M. xanthus BS248 in sterile soil amended with rabbit manure (1:1) increased up to five weeks compared to one week in soil without the manure, suggesting that organic amendment could enrich myxobacteria in soil.

1. Introduction

It is estimated that globally 20–30% of the agricultural yield is lost due to diseases, pests, and the ancillary effects of various measures used to control their actions [1,2]. Fungal diseases are among the leading causes of crop diseases, representing 70–80% of all such diseases [3]. The three species of fungi and oomycetes used in this study cause different diseases in different crops. Rhizoctonia solani is a soilborne pathogen of the division Basidiomycota that causes foliar blight, root rot, seed rot, pre- and post-emergence damping off, and hypocotyl rot in a wide range of hosts, with a worldwide distribution [4]. Sclerotinia sclerotiorum, also known as white mold, is of the division Ascomycota and can infect over 400 plant species from a wide range of families [5]. The stem rot caused by Sclerotinia can lead to devastating economic losses in soybean [6]. Pythium ultimum, the third pathogen used in this study, belongs to the peronosporalean lineage of oomycetes. It causes damping off and root rot diseases in hundreds of diverse plant hosts, including maize, soybean, potato, wheat, fir, and numerous ornamental species [7].

While chemical-based fungicides and pesticides are effective at combating plant diseases and, in many instances, necessary, they can have adverse effects on critical natural environmental processes, including soil microbial activities, and increase weed-resistant populations, soil compaction, and water pollution, which seriously affect sustainable agriculture [3,8]. Thus, biocontrol using antagonistic microorganisms is seen as an essential alternative strategy for achieving sustainable agriculture within an integrated management program, which includes developing better disease-resistant varieties [8].

Extensive research aimed at discovering biocontrol agents and the mechanisms underlying their activities has led to the development of a wide range of products that are now marketed and available worldwide. Species of Trichoderma are among the most widely used microorganisms in such commercial biocontrol products [9]. Myxobacteria have been known to inhabit soil at concentrations of 10³–10⁵ cells/g of topsoil [10], and they possess an inherent ability to prey on and kill microorganisms. This group of bacteria are known as producers of secondary metabolites and enzymes that are used to kill and prey on other microorganisms [11]. The effectiveness of myxobacteria against fungi, bacteria, and oomycetes has been explored previously [12,13,14]. Furthermore, a strain of Corallococcus was formulated as both liquid and solid preparations, which were effective in both the greenhouse and field against cucumber Fusarium wilt [15]. Similarly, a strain of Myxococcus was found to be an effective biocontrol agent against bacterial soft rot of Calla lily (Zantedeschia spp.) caused by Pectobacterium carotovorum [13]. These investigations clearly demonstrated that Myxococcus strains have potential use as plant-disease biocontrol agents.

In this study, we examined the inhibitory activity of 13 strains of myxobacteria on four common crop pathogens using modified methods for the challenge study and a new method for the enumeration of myxobacteria. A mixture of two myxobacterial strains was used as an inoculum to control Rhizoctonia solani against cucumber seedlings in pots in growth chambers. The survival of one myxobacterial strain in soil with various ratios of rabbit manure was evaluated. We also discuss the possibility of using these bacteria as biocontrol agents against fungal diseases in support of sustainable agriculture goals and practices.

2. Materials and Methods

2.1. Myxobacterial and Fungal Strains

The myxobacterial strains Cystobacter fuscus (ATCC 25194), Myxococcus flavus (ATCC 29617), M. flavescens (ATCC 51243), M. virescens (ATCC 29616), Polyanion brachysporum (ATCC 53080), and Corallococcus (Myxococcus) coralloides (ATCC 25202) were purchased from the ATCC and propagated according to the supplier’s directions. In addition to the ATCC cultures, the strains M. xanthus BS 245, BS 248, DK 801, DK 836, and DK 897; M. stipitatus BS 247; and M. coralloides BS 249 were available from Dilip Lakshman’s laboratory at the USDA-ARS, Agricultural Research Service, Beltsville, Maryland. These strains were obtained from other collaborators and preserved in 1 mL cryogenic vials in 20% glycerol at −80 °C and are referred to collectively as DL strains. All cultures were grown and maintained on Casitone yeast extract agar (CYEA). The CYEA composition per liter consisted of Casitone (Life Technologies, Detroit, MI, USA) 10 g, yeast extract (Life Technologies Corp., Detroit, MI, USA) 1 g, calcium chloride (St. Louis, MO, USA) 1 g, and agar (Life Technologies Corp., Detroit, MI, USA) 15 g [16].

The CYEA medium supports good growth for all of the myxobacterial strains and fungal isolates. An incubation temperature of 27.5 °C was chosen, as this is the midpoint for the optimum growth of bacteria (30 °C) and fungal strains (25 °C).

The plant pathogenic isolates used in this investigation were R. solani AG4 (isolate DL 019), R. solani AG2 2LP (DL 058), Sclerotinia sclerotiorum (DL 044), and Pythium ultimum (DL047). Cultures of these four pathogens were grown on Potato Dextrose Agar (PDA, Difco Laboratories, Detroit, MI, USA) for 7 days and used as inoculum. All four of these strains are known to cause various diseases in a range of crops.

2.2. Fungal and Oomycete Growth Rate

The growth rates of all four plant pathogen strains used in this study were determined on Petri dishes (100 × 15 mm) containing 20 mL of CYEA. A 5 mm plug of fungal isolates from actively growing cultures on plates containing PDA was obtained using a sterilized metal cork-borer and placed at the center of the plates with the fungal growth facing the medium. For each strain, five plates were prepared and incubated at 27.5 °C. The radial growth in millimeters was measured four days per week. The average radial growth values with the standard of error for each of the four phytopathogens at various incubation times were calculated using the tool provided at https://www.allmath.com/standard-error-calculator.php (accessed on 4 August 2025). The data were tabulated.

2.3. In Vitro Inhibition of Pathogens by Myxobacterial Strains

Inhibition of the four fungi and oomycete strains by the myxobacterial strains was measured on CYEA medium (20 mL in 100 × 15 mm Petri dishes). Myxobacteria, actively growing for 3 days, were aseptically scraped from the CYEA media and suspended in 0.5 mL of sterile water and vortexed to achieve a uniform cell suspension for use as inoculum. The latter was spread on the surface of a new CYEA agar plate in the form of a 50 mm diameter ring using a sterile loop. A visual template positioned on the exterior bottom of the Petri dishes served as a guide to position the circular inoculum ring. The plates were handled gently and allowed to absorb the liquid inoculum suspension; thereafter, they were incubated at 27.5 °C. When the bacterial growth ring appeared after 3–5 days, a 0.5 cm diameter plug of the actively growing fungus or oomycete (5 days of growth on PDA) was placed in the center of the ring on the agar’s surface. For each bacterial strain and fungal or oomycete isolate combination, five plates were prepared. Fungal radial growth was measured in mm from the edge of the pathogen plug to the farthest radius of growth after 1, 2, 7, and 14 days. Fungus or oomycete growing on CYEA medium without a myxobacterial ring was considered as the control. A 14-day duration for the trial was selected, because by 14 days the radial growth on the control plates had completely covered the agar surface for most of the pathogens. Also, 14 days is sufficient time for the fungal or oomycete pathogens to grow and cross the bacterial barrier, if they could do so. In each challenge of a pathogen vs. a myxobacterial strain, the radial growth on all five treatment plates and the control plates for each incubation period were averaged and used for calculation of the percent inhibition according to the equation below:

% Inhibition = [1 − radial growth (mm) in the presence of a bacterial ring/radial growth (mm) on the control plate] × 100.

Each pathogen and myxobacterial strain challenge trial was repeated, and the percentage inhibition values of the two trials were averaged, and the results were tabulated.

2.4. Inhibition of Rhizoctonia by Myxobacteria in Pots

Cucumber seedlings were propagated from seeds (Heirloom organic, Purely Organic Marketmore 76, SimplyGro LLC, Manchester, NH, USA) germinated in 1.9 L pots containing Pro-Mix potting soil (Quakertown, PA, USA). Seedlings were 20 days old with four leaves when used.

The Rhizoctonia inoculum used in the experiment was prepared according to the method described as follows: Mycelia from two plates each for R. solani AG 4 DL 019 and R. solani AG2 DL 058 covering the entire surface of the PDA in the Petri dishes (100 × 15 mm) were harvested using sterile scalpels, added to 100 mL of sterile distilled water, and then blended in a sterile standard food-grade blender for 1 min.

Myxobacterial inoculum was prepared by scraping the surface of 5 plates each of strains BS 249 and BS 248 grown separately on CYEA medium for four days. The scraped bacteria were added to 100 mL of sterile distilled water and blended for 1 min, as described above for the Rhizoctonia inoculum preparation.

The experiment began as follows: 2 mL of Rhizoctonia inoculum prepared as described above was added to containers (volume of about 400 mL) filled with moistened potting soil Pro-Mix (Quakertown, PA, USA). The inoculum was mixed with the soil using a spatula and left overnight in the laboratory for the Rhizoctonia to initiate growth. Rhizoctonia was not added to the pots designated as controls. After 24 h, two seedlings, prepared as described above, were transplanted into the containers. After transplanting, an inoculum of myxobacteria was added to the pots to prepare four different treatments, as indicated in

Table 1. The scheme for the addition of Rhizoctonia and myxobacteria to soil in the pots for the four different treatments.

Treatment	Rhizoctonia	Myxobacteria
1 Positive control	No	No
2 Negative control	Yes	No
3	Yes	Yes, 4 mL
4	Yes	Yes, 8 mL

below.

For each of the four treatments, two pots with two seedlings each (totaling 4 seedlings per treatment) were prepared and incubated in a growth chamber (Conviron, Controlled Environments Inc., Pembina, ND, USA) set at 27 °C and 50% relative humidity, with a 12 h. alternating light/dark cycle. Seedlings were monitored for growth and signs of disease, and they were watered every other day with 30 mL distilled water. After two weeks, the pots were photographed, the three largest leaves from each treatment were cut, and the dimensions (width and height) were measured in mm. The leaf dimensions were analyzed statistically using ANOVA and the Tukey HSD test, at p = 0.05, using an online tool available at https://www.socscistatistics.com/tests/anova/default2.aspx (accessed on 10 June 2025).

2.5. Survival of Myxobacteria Strain BS 248 in Soil Amended with Rabbit Waste

Rabbit manure purchased from HDH Company, St. Louis, MO, USA, was ground in a blender into fine particles and mixed with soil obtained from corn–soybean-rotated fields at the Beltsville Agricultural Research Center (10300 Baltimore Avenue, Beltsville, MD, USA) to obtain concentrations of (w/w) 0, 25, 50, 75, and 100% manure in soil. The soil and rabbit manure mixture (total weight: 10 g) was mixed in flasks and autoclaved for 30 min; autoclaving was repeated twice on consecutive days. Thereafter, the mixture was aseptically transferred to 50 mL sterile Falcon tubes. Sterile deionized water was added to the mixture to moisten but without excess standing liquid. Duplicate tubes were used for each combination of soil and rabbit fecal pellets. Inoculum for the experiment was prepared by scraping biomass, using a sterile inoculation loop, from three days’ growth of M. xanthus BS248 on CYEA medium in a single 100 × 15 mm Petri plate, transferred to 10 mL of sterile saline solution, and vortexed to a uniform suspension, from which 1 mL was added to each tube containing the soil–manure mixture. The bacterial count of the inoculum was determined using serial dilutions of the suspension (10⁻¹–10⁻⁶) performed in tubes containing 9 mL of sterile 0.9% saline solution, from which 0.1 mL of the dilutions was distributed on two plates of CYEA medium as 20 drops of 5 µL each, since spreading of inoculum usually appears as a mass of growth without distinction of individual colonies. Similarly, the myxobacterial strain in the soil mixture was measured weekly by aseptically transferring 1 g of the mixture into 9 mL of sterile saline solution for a dilution of 10⁻¹. From the latter, serial dilutions of 10⁻²–10⁻⁵ were prepared, and 0.1 mL from each dilution was added to two plates of CYEA as 20 drops of 5 µL. Plates were incubated at 27.5 °C and checked for two weeks for the presence of typical colonies of myxobacteria. The dilution with the highest number of bacteria/g was recorded.

3. Results

3.1. Determination of the Growth Rate for the Phytopathogens Used in the Study

The fungal and oomycete species that were chosen for the challenge study had different growth rates. The growth rates of the four species were determined on CYEA medium at 27.5 °C. The isolates R. solani DL 019 and DL 058 had high growth rates and filled the plates (47.5 mm of radial growth) in three and six days, respectively (

Table 2. Radial growth of the four pathogens on CYEA medium in Petri dishes (100 × 15 mm) incubated at 27 °C.

	Incubation Time (Days)
Phytopathogen	1	2	3	6
R. solani AG4 DL 019	10.25 ± 0.48	22.75 ± 0.25	32.5 ± 0.29	F *
Sclerotinia sclerotiorum DL 044	5.00 ± 0	8.75 ± 0.25	12.75 ± 0.25	18.75 ± 0.63
Pythium ultimum DL 047	5.25 ± 0.25	12.5 ± 0.29	20.75 ± 0.48	31.25 ± 6.25
R. solani AG2 DL 058	17.25 ± 0.63	32.75 ± 0.45	F	F

* Petri dish was fully covered with pathogen growth.

). However, P. ultimum DL 047 and S. sclerotiorum DL 044 grew at a slower rate than the R. solani strains. Their radial growth after six days reached approximately 19 and 31 mm, respectively.

3.2. In Vitro Inhibition of Four Phytopathogens by 13 Myxobacterial Strains

The inhibition of R. solani AG 4 strain DL 019 by 13 myxobacterial strains on CYEA medium is shown in

Table 3. Percent inhibition of four phytopathogens by 13 myxobacterial strains on CYEA media incubated at 27.5 °C.

Pathogens	Incubation Time	Myxobacterial Strains
	Days	* A	B	C	D	E	F	G	H	I	J	K	L	M
Rhizoctonia solani	1	60	93	80	100	85	61	68	12	14	26	24	49	27
AG4	2	73	88	73	99	86	53	85	29	52	60	60	74	60
DL 019	7	66	86	72	95	84	44	93	0	0	0	20	84	48
	14	76	86	55	90	85	49	93	0	0	0	0	85	48
Sclerotinia	1	30	100	94	100	100	50	40	0	34	54	50	50	75
sclerotiorum	2	57	91	88	100	100	60	46	26	49	65	100	100	83
DL 044	7	79	95	93	80	100	35	81	72	67	88	100	100	92
	14	78	93	100	75	100	50	84	47	65	76	100	100	93
Pythium	1	16	50	33	93	11	66	0	28	34	34	34	34	22
ultimum	2	15	35	13	93	39	79	24	11	11	26	28	40	36
DL 047	7	53	56	57	72	46	65	64	42	29	22	60	64	53
	14	55	56	55	60	33	0	53	36	20	24	60	56	46
Rhizoctonia solani	1	71	84	68	81	74	36	79	8	16	44	40	88	16
AG2	2	66	83	52	90	78	54	96	37	45	60	29	92	38
DL 058	7	68	81	56	88	82	56	96	0	0	50	73	91	65
	14	62	81	56	78	84	0	97	0	0	0	71	94	59

* A, Cystobacter fuscus ATCC 25194; B, Myxococcus flavus ATCC 29617; C, M. flavescens ATCC 51243; D, M. virescens ATCC 29616; E, Polynion brachysporum ATCC 53080; F, M. (Corallococcus) coralloides ATCC25202; G, Myxococcus xanthus (Mx) BS 248; H, Mx DK 836; I, Mx BS 245; J, M. stipitatus BS 247; K, Mx DK 897; L, M. (Corallococcus) coralloides BS 249; M, Mx DK 801.

. After two weeks of incubation, two myxobacterial strains—M. virescens ATCC 29616 and Mx BS 248—exhibited the greatest inhibition, at 90% and 93%, respectively. In contrast, the M. xanthus strains DK 836, BS 245, and DK 897 and M. stipitatus BS 247 provided no inhibition, as the fungus was able to cross the bacterial line, which indicates zero inhibition. Figure 1 illustrates the inhibition of R. solani DL 019 by strain M. xanthus BS 248 on the CYEA plate. Significantly greater radial growth can be observed on the control plate than on the treatment plate. There is a clear zone between the fungal plug in the middle and the myxobacterial growth line, indicating that the fungus was inhibited without any contact with the bacterial growth line.

Against the fungal species S. sclerotiorum strain DL 044, in general, the myxobacterial strains were highly effective growth inhibitors (Table 3). In total, four strains provided total inhibition of the pathogen, as follows: M. flavescens ATCC 512343, P. brachysporum ATCC 53080, and M. coralloides BS 249 and Mx DK 897. Furthermore, two strains, Myxococcus flavus ATCC 29617 and Mx DK 801, each provided 93% inhibition. Meanwhile, the strain with the lowest inhibitory effect was C. coralloides ATCC 25202, which provided 50% inhibition. Unlike R. solani DL 019, this pathogen was unable to cross the bacterial line in any of the 13 challenges. Figure 2 shows the inhibition of the pathogen S. sclerotiorum DL 044 by the myxobacterial strain Myxococcus coralloides BS 249. The radial growth on the control plates, as shown by arrow C, is much greater than the very limited or no growth on the treatment plate, as indicated by arrow B. This is an example of 100% inhibition. Like the inhibition of R. solani DL 019 shown in Figure 1, the inhibition of S. sclerotiorum DL 044 occurred without any contact with the bacterial growth line.

The inhibition exhibited by the myxobacterial strains against Pythium ultimum DL 047 was moderate and did not exceed 60% (Table 3) Among the ATCC strains, Cystobacter fuscus ATCC 25194, Myxococcus flavus ATCC 29617, M. flavescens ATCC 51243, and M. virescens ATCC 29616 provided moderate inhibition rates of 55–60%. The inhibition exhibited by strain P. brachysporum ATCC 53080 was 33%, while the inhibition by M. coralloides ATCC 25202 was zero after the oomycete species crossed the bacterial line during the two-week incubation period. Among the laboratory strains Mx BS 249, Mx DK 897, and M. coralloides, BS 249 provided moderate inhibition between 50 and 60%. Three other myxobacterial strains produced inhibition levels below 50%. The isolates M. xanthus BS 245 and Myxococcus stipitatus BS 247 were the least effective inhibitors of the pathogen, with 20% and 24% inhibition rates, respectively.

Figure 3 shows the inhibition of P. ultimum DL 047 by the myxobacterial strain BS 249. The figure shows that the radial growth was greater on the control plate than on the treatment plate, as indicated by the length of arrows B and C. There is no clear zone between the pathogen and the bacterial line. The growth of P. ultimum was slowed upon contact of the oomycete with bacterial growth line A.

Against R. solani AG2 strain number DL 058 and (2LP), four strains of myxobacteria—isolates Myxococcus flavus ATCC 29617, P. brachysporum ATCC 53080, Mx BS 248, and M. coralloides BS 249—were the most effective strains, providing inhibition levels above 80% (Table 3). In contrast, four strains of myxobacteria provided zero inhibition, as the fungus crossed the bacterial line of the myxobacterial strains. These strains were M. (Corallococcus) coralloides ATCC25202, Mx DK 836, Mx BS 245, and M. stipitatus BS 247. These strains are the same strains that were ineffective against the other R. solani strain DL 019. Figure 4 illustrates the inhibition of the fungus by strain M. xanthus BS248 after two weeks of incubation. As can be observed, the fungus was strongly inhibited, and there is a clear zone between the fungus plug and the bacterial growth line. The fungus in the control plate, however, grew well, covering the entire plate.

In summary, among all 13 myxobacterial strains, the M. xanthus BS 248, M. coralloides BS249, and M. flavus ATCC 29617 designations, shown in

Table 4. Percent inhibition of four phytopathogens by 13 strains of myxobacteria on CYEA medium in Petri dishes incubated at 27.5 °C for two weeks.

Pathogen Strain	Myxobacterial Strains
	A a	B	C	D	E	F	G	H	I	J	K	L	M
DL 019 b	76	86	55	90	85	49	93	0	0	0	0	85	48
DL 044	78	93	100	75	100	50	84	47	65	76	100	100	93
DL 047	55	56	55	60	33	0	53	36	20	24	60	56	46
DL 058	62	81	56	78	84	0	97	0	0	0	71	94	59

^a A, Cystobacter fuscus ATCC 25194; B, Myxococcus flavus ATCC 29617; C, M. flavescens ATCC 51243; D, M. virescens ATCC 29616; E, Polynion brachysporum ATCC 53080; F, M. (Corallococcus) coralloides ATCC25202; G, Myxococcus xanthus (Mx) BS 248; H, Mx DK 836; I, Mx BS 245; J, M. stipitatus BS 247; K, Mx DK 897; L, M. (Corallococcus) coralloides BS 249; M, Mx DK 801. ^b DL 019, Rhizoctonia solani AG4; DL 044, Sclerotinia sclerotiorum; DL 047, Pythium ultimum; and DL 058, R. solani AG2.

, B, G and L, which correspond to M. xanthus BS 248, M. coralloides BS249, and M. flavus ATCC 29617, respectively, were the most effective inhibitors of all four plant pathogens used in this study. These myxobacterial strains can be categorized as strong inhibitors, whereas strains Corallococcus coralloides ATCC 215202 and Myxococcus xanthus BS 245, designated as F and I, respectively, could be considered weak inhibitors, as they provided the least inhibition against all of the pathogens. The remaining strains are categorized as moderate inhibitors. The bacterial strains exerted inhibitory action both by contact and non-contact against the pathogens.

3.3. Inhibition of Rhizoctonia Effects on Cucumber Seedlings by Myxobacteria

The effect of myxobacteria on cucumber seedlings challenged with Rhizoctonia species was profound. The seedlings grown in the presence of Rhizoctonia were stunted and had smaller leaves with more yellowish color, as shown in Figure 5A,B and Figure 6, treatment 2 (T2). However, the effect of Rhizoctonia was diminished in T3 and T4 when the soil was inoculated with myxobacteria to neutralize the pathogen. Clearly, plants in T3 and T4 were healthier, bigger, and had large leaves compared to seedlings grown in soil with Rhizoctonia but without myxobacteria (T2). The average widths of the three largest leaves from treatments 1–4 were 50 ± 4.3, 39.3 ± 0.58, 63.0 ± 3.6, and 60.0 ± 5.0, respectively. The average heights of the leaves were 44.6 ± 1.5, 34.6 ± 1.5, 48.7 ± 3.7, and 46.7 ± 4.5, respectively. The statistical analysis via ANOVA of the dimensions of the leaves indicates that the leaves from T2 were significantly smaller (p-value of 0.05) than those in the other three treatments. However, there were no differences in the leaf dimensions from T1, T3, and T4. The addition of 4 or 8 mL of the myxobacterial inoculum had similar effects, as there was no statistical difference in the sizes of the leaves from the plants in T3 and T4. The leaf dimensions mentioned above and the health of the plants (Figure 5A,B) show that the plants in T3 and T4 had bigger leaves and were healthier than in T1 (seedlings grown in the absence of Rhizoctonia and myxobacteria). However, the differences in the size of the leaves were not statistically significant, with the p-values for T1 vs. T3 and T1 vs. T4 being 0.12 and 0.36, respectively.

3.4. Effect of Rabbit Manure in the Soil on the Survival of Myxococcus xanthus

In the survival study, the effect of supplementing sterile soil with different concentrations of rabbit manure showed that in the mixture of 50% manure in soil, M. xanthus BS 284 survived for 5 weeks compared to one week in soil without manure supplementation (

Table 5. Survival of Myxococcus xanthus strain BS 248 in sterile soil amended with different concentrations of rabbit manure.

Percent Rabbit Manure in Soil
Incubation Time (Weeks)	0%	25%	50%	75%	100%
1	4 × 106 a	2 × 106	7 × 106	2 × 107	2 × 106
2	ND b	3 × 105	2 × 107	8 × 106	ND
3	ND	ND	1 × 106	1.4 × 104	ND
5	ND	ND	2 × 104	ND	ND

^a cfu/g of the soil–manure mixture. ^b M. xanthus typical colonies were non-detectable; minimum detectable was 10 CFU/g.

). The results also show that the bacterial number did not increase substantially in any of them and only slightly in the 50% manure. The number of M. xanthus remained near the inoculum level (1 × 10⁷) at best. The mixtures of 75% and 25% manure in soil increased the survival of the myxobacterial strain to 3 and 2 weeks, respectively.

4. Discussion

Myxobacteria are soil-inhabiting, Gram-negative, and rod-shaped bacteria. Based on their feeding habits, they comprise the following two groups: (a) bacteriolytic, which feed on microorganisms, and (b) cellulolytic, which hydrolyze cellulose and feed on dead cells. The majority of cultured myxobacteria are bacteriolytic, and, thus, the group has biocontrol application potential [17]. In this investigation, the inhibitory effect of 13 myxobacterial strains belonging to four genera was tested against four strains of three different plant pathogens. Three of the pathogens were fungal species, and one belonged to oomycetes, a distinct phylogenetic lineage of fungus like eukaryotic microorganisms. These species are among the most common crop pathogens and together with a few other common pathogens, such as Fusarium, Phytophthora, and Verticillium, can cause yield losses of up to 75% in cereals, cotton, and horticultural vegetables [18]. Different inhibition levels were shown by the strains of myxobacteria. Three strains, all belonging to the genus Myxococcus (M. xanthus BS 248, M. coralloides BS249, and M. flavus ATCC 29617), were placed in the high-inhibitory strain category for their high inhibition of all of the phytopathogens. This genus is the most frequently occurring around the world in soil with a pH range of 5–8 [10]. It is essential to note that within Myxobacteria, the ability to produce certain inhibitory compounds is typically a strain characteristic, rather than a species characteristic. This explains why some of the strains of M. xanthus were highly inhibitory to the pathogens, whereas others were not [16].

The growth rate of the pathogens was an essential factor determining the inhibitory effect. In general, the myxobacterial strains were more potent against slow-growing pathogens than against fast-growing ones. All 13 strains of myxobacteria tested were less inhibitory toward P. ultimum DL 047 than S. sclerotiorum DL 044; the two pathogens differed in growth rate, with DL 047 exhibiting a higher rate than DL 044. Also, in the 26 challenges between the two high-growth-rate fungi, R. solani strains DL 019 and DL 058, and the 13 myxobacteria strains, in 8 out of those 26 challenges, the inhibition levels dropped to zero after two weeks of incubation, as the pathogen crossed the bacterial growth line. However, crossing of the bacterial line happened only in 1 case out of the 26 challenges between the two slow-growing pathogens P. ultimum DL 047 and S. sclerotiorum DL 044 and the 13 myxobacterial strains. In agreement with this study, it has been reported that species of the genera Pythium and Sclerotinia were the most sensitive to the effect of myxobacteria, while the fast-growing fungi such as Fusarium and Verticillium were resistant to the myxobacterial inhibitory effects [12]. It is noteworthy to report that in [12], the plates in the challenge study were incubated at room temperature, which is not a definitive statement. Therefore, the temperature could have favored either the pathogen or the predatory bacteria. In this study, however, the incubation temperature of 27.5 °C is equally favorable to the pathogens and myxobacteria, as it is the midpoint between the optimum growth temperature of 25 °C and 30 °C for the pathogens and bacteria, respectively.

The results reported here shed light on some aspects of the mode of action of myxobacterial strains on the pathogen species. Against the three fungal species, as shown in Figure 1, Figure 2 and Figure 4, we observed that the inhibition of the fungi did not require any contact with bacterial growth, as there is a clear zone between the fungal plug and the bacterial growth lines. However, against the oomycete species P. ultimum (Figure 3) there was no clear zone between the fungal inoculum plug and the bacterial growth. Growth of the pathogen was stopped upon contact with the bacterial growth line. Myxobacteria are known to produce a large number of metabolites with unique structures [19]. Low-molecular-weight biomolecules will diffuse faster than lytic enzymes in media and will lead to inhibition or lysis of fungal cells without any contact. Myxovirecin, an antibiotic produced by M. xanthus, was proven responsible for the inhibition of Escherichia coli based on studies with a mutant of M. xanthus defective at producing the antibiotic [20].

Regarding the inhibition upon direct physical contact, it can be explained by the presence of high-molecular-weight, less mobile lytic enzymes that remain near the bacterial line, thereby lysing a pathogen upon direct physical contact. The difference in cell-wall components of fungi and oomycete could reflect the difference in the way the myxobacterial strains exert their inhibition. The main component of fungal cell walls is chitin, whereas cellulose is the predominant component of cell walls of oomycetes. The lytic enzymes β-1,6-gluconase, β-1,3-gluconase, chitinase, protease, and peptidase are the main enzymes that participate in cell predation [19].

There have been reports of inhibitory effects of volatile compounds produced by myxobacteria. The myxobacterial strain “Corrallococcus sp. EGB” emits volatile organic compounds (VOCs) that have a significant inhibitory effect on various fungal plant pathogens, demonstrating potential as a biocontrol agent against fungal plant diseases [21]. However, the cultures we used had no discernible smell or odor and, thus, it is possible that VOCs had no impact on the inhibition rates in these challenge studies.

When tested in pots against R. solani isolates DL 019 and DL 058, a mixture of two strains, M. xanthus BS 248 and M. coralloides BS249, prevented the effect of the pathogens in cucumber. Additionally, the plants in pots amended with myxobacteria were larger and greener with bigger leaves than plants grown in pots not inoculated with Rhizoctonia and myxobacteria (controls), suggesting possible growth-promoting abilities of some myxobacterial strains. We are unaware of previous reports of growth promotion by myxobacteria. However, some strains of other biocontrol agents like Trichoderma have been reported as providing growth-promotion activities [22].

During this study, it was observed that the myxobacterial strains did not survive well on the plates. In general, the cultures on CYEA medium needed to be transferred to new plates within 10 days to maintain the cultures in a viable state. Also, the strains of myxobacteria did not grow well in liquid media. Thus, we emphasize that the viability of myxobacteria will be a critical factor in using these microorganisms in products for amending soil for biocontrol. Another possible way to take advantage of the inhibitory capacity of these bacteria against pathogenic fungi, as well as other types of pathogens, is to manage soil conditions that favor enrichment of these myxobacteria in soil specifically and increase microbial diversity in general. Reduction in soil microbial diversity has been implicated in increased soilborne pathogens [23]. In an extensive field study, the results showed that the addition of swine manure to the soil increased the abundance and density of myxobacteria in the soil. In contrast, nitrogen fertilizers decreased both the abundance and density of this group of bacteria [24]. These results agree with the findings of the study reported here, that soil amended with rabbit manure increased the survival of a species of myxobacteria compared to non-amended soil. Our results also show that the myxobacteria did not increase significantly in the soil during incubation, suggesting that myxobacterium M. xanthus strain BS 248 may prefer to obtain the required nutrients by preying on other microorganisms rather than from media or organic matter in the soil. This could be the reason for the poor growth and survival of these bacteria in typical laboratory media.

5. Conclusions

Significant inhibition of pathogenic fungi and oomycetes by myxobacteria was observed in laboratory tests in Petri dishes and in soil pots. Larger-scale tests in greenhouses or fields are needed to test the effectiveness of these bacterial strains against specific pathogens and in particular crops. Moreover, tests against beneficial organisms such as Trichoderma species and other useful bacteria are essential to conduct, even though, based on our results, it is expected that fast-growing fungi like Trichoderma sp. will not be inhibited by myxobacteria. Based on the difficulties encountered in maintaining and growing myxobacteria and the fact that rabbit manure (in this study) increases the survival of myxobacteria, we suggest further tests for the amendment of soil with composts to enrich myxobacteria specifically for increased suppression of soilborne fungal phytopathogens.