M.H. Ryder

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

ABSTRACT Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum andPseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.

Use of strains of Bacillus isolated in China to suppress take-all and rhizoctonia root rot, and promote seedling growth of glasshouse-grown wheat in Australian soils

Abstract Several strains of Bacillus subtilis and B . cereus , isolated and selected in China for their ability to promote plant growth and control root disease (yield-increasing bacteria), were tested for their potential to control take-all, caused by Gaeumannomyces graminis var. tritici , and rhizoctonia root rot caused by R . solani AG-8 on wheat seedlings grown in field soils in a glasshouse. Bacillus cereus isolate A47 and B . subtilis B908 consistently reduced the severity of take-all of wheat grown in a sodic acid soil. The amount of disease control was similar to that obtained with the biocontrol isolate Pseudomonas corrugata 2140. B . subtilis B931 was considerably more effective than other bacterial treatments in reducing the severity of rhizoctonia root rot in a calcareous sandy loam soil. Control of rhizoctonia root rot with isolate B931, and also with B . cereus strains A47 and M22 was both substantial and reproducible. Seedling growth promotion effects following bacterial treatment were less consistent than disease control effects. B . subtilis B908 and B . cereus A47-2 and A47-3 gave the most frequent positive growth responses (wheat seedling root weight, shoot weight and shoot length) in the absence of pathogen inoculum. This is the first reported research outside China where these Bacillus isolates have given control of soil-borne root diseases and promoted growth of healthy seedlings under glasshouse conditions.

Biological control of take-all of glasshouse-grown wheat using strains of Pseudomonas corrugata isolated from wheat field soil.

Abstract Bacteria were isolated from the rhizospheres of wheat plants. Selected bacterial isolates, some inhibitory and some non-inhibitory in vitro to Gaeumannmyces graminis var. tritici (Ggt), were tested for their ability to control take-all on wheat in a pot assay. The isolates were assayed in a natural (non-sterilized) soil-sand mixture with added Ggt inoculum. A group of non-fluorescent Pseudomonas isolates gave significant and reproducible disease suppression. One of these isolates stimulated plant growth in the absence of disease, though this effect was not consistent. The non-fluorescent isolates were identified as most closely related to Pseudomonas corrugata. Strain 2140 of Pseudomonas corrugata controlled take-all in vivo at 15°C and inhibited the growth of Ggt in vitro in the range 10–25°C.

Suppression of damping-off caused by Rhizoctonia solani, and growth promotion, in bedding plants by binucleate Rhizoctonia spp

Abstract Nine isolates of binucleate Rhizoctonia spp, from potting media and nursery plants, were screened for their ability to control damping-off disease caused by Rhizoctonia solani Kuhn AG 4 and AG 8. Seedlings of Capsicum annuum L. were grown in a pasteurized potting medium in growth chambers. All isolates prevented damping-off caused by R. solani AG 4, and most isolates increased shoot weights in the presence of either AG 4 or AG 8. Two isolates were tested further on seedlings grown in pasteurized potting medium in a glasshouse. In two experiments, both isolates controlled damping-off in Capsicum caused by AG 4. In another experiment, both isolates reduced damping-off caused by AG 4 in Capsicum, Celosia and Viola seedlings, and increased shoot weights of Capsicum and Celosia. One isolate also reduced damping-off in Petunia. Each isolate promoted shoot growth of seedlings in two of four experiments when no pathogen was added. As these binucleate Rhizoctonia isolates also control Pythium, they have potential for use as biological control agents in plant nurseries.

Ability of the lumbricid earthworms Aporrectodea rosea and Aporrectodea trapezoides to reduce the severity of take-all under greenhouse and field conditions

Abstract The influence of the earthworms A. rosea and A. trapezoides on wheat plants, grown in soil artificially infested with the take-all fungus Gaeumannomyces graminis var. tritici (Ggt) was examined. In pot trials, using a red-brown earth soil artificially infested with Ggt , the presence of the earthworm A. trapezoides (at a density equivalent to 314 or 471 m −2 ) was associated with a significant ( P Ggt , shoot weight was not significantly ( P > 0.05) influenced by the presence of A. trapezoides .Two field trials were conducted, in which A. rosea and A. trapezoides were added, at an equivalent density of 100 or 300 m −2 , to cylinders driven into the soil. In a calcareous sandy loam artificially infested with Ggt , the presence of the earthworms A. rosea or A. trapezoides (at these densities) was associated with a significant ( P A. rosea or A. trapezoides (at an equivalent density of 300 m −2 ) caused a significant increase in shoot weight. In contrast, in the absence of added Ggt , shoot weight was not significantly ( P >0.05) influenced by the presence of A. rosea or A. trapezoides . In a second field trial in a red-brown earth, the presence of the earthworm A. trapezoides (at an equivalent density of 300 m −2 ) was associated with a significant ( P A. rosea or A. trapezoides did not cause a significant increase in shoot weight. In the absence of Ggt , shoot weight was not significantly ( P >.05) influenced by the presence of A. rosea or A. trapezoides .These results demonstrate the potential of the earthworms A. rosea and A. trapezoides , under both greenhouse and field conditions, to reduce the severity of take-all disease on wheat.

Reduced severity of rhizoctonia solani disease on wheat seedlings associated with the presence of the earthworm aporrectodea trapezoides (lumbricidae)

Abstract The influence of the earthworm Aporrectodea trapezoides on wheat plants grown in a calcareous sand loam and a red-brown earth soil, artificially infested with Rhizoctonia solani , was examined. Presence of the earthworm A. trapezoides , at a number equivalent to 471 m −2 was associated with a significant increase in shoot weight and a reduction in the root disease rating of wheat in both soil types artificially infested with R. solani on wheat chaff. In both soil types, the shoot weight of wheat grown in the presence of A. trapezoides and R. solani was equivalent to that of wheat grown in the absence of R. solani. One further treatment was applied to each soil type. In the calcareous sand inoculated with R. solani on wheat chaff, A. trapezoides was as effective as mechanical soil disturbance in reducing the root disease rating on wheat. In the red brown earth, in which R. solani was introduced via naturally infected roots, the presence of A. trapezoides was associated with a significant increase in shoot weight and a reduction in the root disease rating of wheat. These results demonstrate the potential of the earthworm A. trapezoides to reduce the effect of Rhizoctonia root rot on wheat.

Binucleate Rhizoctonia isolates control damping-off caused by Pythium ultimum var. Sporangiiferum, and promote growth, in Capsicum and Celosia seedlings in pasteurized potting medium

Abstract Eight isolates of binucleate Rhizoctonia spp, from South Australian plant nurseries and potting mix suppliers, were screened for ability to control damping-off disease caused by Pythium ultimum var. sporangiiferum. The screening was by two bioassays on seedlings of Capsicum annuum grown in a pasteurized potting medium in a glasshouse or controlled environment growth chamber. Three isolates reduced damping-off at least as much as the fungicide propamocarb. The fungicides tolclofos-methyl and benomyl did not reduce disease or increase shoot weights. In other glasshouse experiments, binucleate Rhizoctonia isolates BNR1 and BNR2 reduced damping-off in Capsicum seedlings more as doses of each antagonist were increased. In pasteurized potting medium without added pathogens, isolates BNR1 and BNR2 increased dry weights of Capsicum shoots per punnet by 33 and 24%, respectively, compared with only 13% for the bacterium, Bacillus subtilis A 13. Isolates BNR1 and BNR2 completely controlled damping-off in seedlings of Celosia argentea, but propamocarb and B. subtilis did not.

Enhanced root nodulation of subterranean clover (Trifolium subterraneum) byRhizobium leguminosarium biovartrifolii in the presence of the earthwormAporrectodea trapezoides (Lumbricidae)

In a greenhouse study, the effect of the earthwormAporrectodea trapezoides on root nodulation in seedlings of subterranean clover (Trifolium subterraneum) was examined in the presence and absence of addedRhizobium leguminosarium biovartrifolii (strain NA 30). WhenR. trifolii NA 30 was inoculated into dung and placed on the soil surface, the total number of root nodules was five times greater (P<0.001) in the presence of earthworms than without earthworms and the number of nodules on the primary root of the plants 2–8 cm below the soil surface was 4 to 6 times greater (P<0.001) in the presence of earthworms. The additional nodulation did not affect plant growth or foliar N. When NA30 was dispersed through the soil at the beginning of the experiment, the presence of earthworms did not influence the level of root nodulation. The presence of earthworms increased root dry weight by 20–30%, plant top weight by up to 125% (P<0.001), and foliar N by 5–25% (P<0.001). Surface-applied dung increased the dry weight of plant tops (2-to 3-fold,P<0.001) but did not affect the concentration of foliar N (P<0.005).

Suppression of Rhizoctonia solani AG-8 induced disease on wheat by the interaction between Pantoea, Exiguobacterium, and Microbacteria

Rhizoctonia solani AG-8 is a major wheat root pathogen; however, soils can become suppressive to the expression of disease under intensive cropping with retention of crop residues. This is in part due to the action of soil microorganisms. A step-wise approach was used to determine which microorganisms contributed to suppression of R. solani induced disease in a disease-suppressive soil. Using wheat-soil-pathogen bioassays it was determined that the interaction between 3 phylogenetically diverse groups of bacteria, Pantoea agglomerans, Exiguobacterium acetylicum, and Microbacteria (family Microbacteriaceae), was a major contributor to disease suppression. Inoculation of a sterilised soil with the combination of these groups resulted in greatly increased seedling shoot dry weight and reduced infection compared with diseased control plants with no bacterial inoculation, or inoculated with individual types of bacteria. These groups, however, did not reduce levels of pathogen DNA, although inoculation with suppressive soil (at 10% w/w) did reduce pathogen DNA. Root associated P. agglomerans and E. acetylicum promoted the growth of infected wheat plants and soil associated Microbacteria reduced root infection by R. solani.

Field evidence for reduced severity of Rhizoctonia bare-patch disease of wheat, due to the presence of the earthworms Aporrectodea Rosea and Aporrectodea Trapezoides

Abstract A study demonstrated the ability of the earthworms Aporrectodea rosea and Aporrectodea trapezoides (added at an equivalent density of 100 or 300 m −2 ) to reduce the disease severity of Rhizoctonia soiani Kuhn on wheat in the field. In a calcareous sandy loam artificially infested with R. solani , the addition of these earthworms caused a significant ( P = 0.02) reduction of the Rhizoctonia root disease rating and had a significant ( P = 0.01) positive effect on shoot weight. Neither earthworm number or earthworm species had a significant ( P R. solani , neither the presence of these earthworms, earthworm species nor earthworm number influenced root disease rating. However, under these conditions the addition of earthworms ( P = 0.01) had a significant positive effect upon shoot weight. To our knowledge these results demonstrate for the first time, the potential of earthworms to contribute to the disease suppression of a cropping soil in a field situation.