Isolation,Identification, and Herbicidal Activity of Metabolites Produced by Pseudomonas aeruginosa CB-4

CB-4, a bacterial strain with highly effective herbicidal activity, was isolated from infected corn leaves. Through morphology, physiological and biochemical tests, and 16 S ribosomal DNA gene sequencing methods, CB-4 was identified as Pseudomonas aeruginosa. We conducted activity-evaluation experiments in the laboratory to assess the herbicidal potential of metabolites produced by strain CB-4. Crude extracts of strain CB-4 have high inhibition activity on Digitaria sanguinalis. In general, the root and shoot growth parameters of D. sanguinalis were significantly reduced by metabolites of strain CB-4. The IC50 of the culture filtrate extracts for the radicula and coleoptile of D. sanguinalis were 0.299 and 0.210 mg mL-1, respectively. Component 2 of the herbicidal activity of the crude toxin from strain CB-4 was successfully purified for the first time by using high-speed counter current chromatography with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water（4：5：4：5, v/v） and high-performance liquid chromatography. We concluded that the metabolites of strain CB-4 have the potential to be developed as a microbe-based herbicide.     

Biocontrol efficacy of Colletotrichum truncatum for hemp sesbania (Sesbania exaltata) is enhanced with unrefined corn oil and surfactant

In greenhouse and field experiments, an oil-in-water emulsion of unrefined corn oil and Silwet L-77 increased the biological weed control efficacy of Colletotrichum truncatum (Schw.) Andrus et Moore for control of the weed, hemp sesbania ( Sesbania exaltata [Raf.] Rydb. ex A.W. Hill). The surfactant – corn oil emulsion stimulated germination and appressoria formation in vivo and in vitro and delayed the need for dew. We hypothesize that the corn oil protected the conidia from desiccation during the dew-free period and the surfactant stimulated spore germination and appressoria formation. In field experiments conducted over 3 years, a single application of a 50% (v/v) unrefined corn oil tank mixture containing 0.2% (v/v) Silwet L-77 surfactant controlled hemp sesbania in soybeans an average of 95%. Aqueous fungal suspensions or adjuvants alone did not visually affect or control hemp sesbania. The soybean yields were significantly higher in the plots where weeds were effectively controlled. These results suggest that formulating C. truncatum in unrefined corn oil and surfactant greatly increases its infectivity and the biocontrol potential of this pathogen.     

Weed-Suppressive Bacteria Applied as a Spray or Seed Mixture Did Not Control Bromus tectorum

We conducted two case studies testing effectiveness of a soil-borne bacteria, Pseudomonas fluorescens strain D7, in controlling Bromus tectorum (cheatgrass) when mixed with native seeds sown after a fire and when sprayed on a native community with high abundances of B. tectorum. Each case study area (162 ha) compared treatments with D7 present and absent and was replicated four times (20.3 ha each) in a completely randomized design. Response variables (foliar cover, aboveground biomass, and density of B. tectorum; density of sown native plants) were measured pretreatment for the sprayed area and each year for 3 yr after treatment at both study areas and were evaluated as a repeated measures analysis. Foliar cover, biomass, and density of B. tectorum with sprayed or seed mixture applications did not differ between D7-treated and untreated areas at any time within the study (F_1,6 ≤ 1.42; P ≥ 0.28). D7 as a seed mixture did not significantly impact densities of native seedlings (F_1,6 = 1.27; P = 0.30) at any time during the study. Results contrasted with previous D7 studies that showed effective control of B. tectorum within 3 yr of treatment. Since bioherbicidal methods are being commonly applied, we believe that reporting negative results is important for future meta-analytical studies that provide managers with information on the likelihood for weed-suppressive bacteria to effectively control weeds.     

The influence of growth conditions on biomass, toxins and pathogenicity of Fusarium oxysporum f. sp. orthoceras, a potential agent for broomrape biocontrol

The influence of different combinations of illumination and shaking on the growth dynamics, pathogenicity and toxin production of the fungus Fusarium oxysporum f. sp. orthoceras, a biocontrol agent of Orobanche cumana, was studied. The fastest biomass accumulation was obtained under shaking, with or without illumination, with the highest biomass obtained after 3–4 weeks of growth. The biological activity of chloroform extracts of the culture filtrate was characterised: it contained at least two main toxic metabolites that caused necrosis and wilting of various plants and led to mortality of germinating seeds of O. cernua, O. aegyptiaca, O. ramosa and O. cumana. The highest toxic activity of the chloroform extract was obtained under illumination without shaking after 3–4 weeks of growth. The two toxic metabolites were purified and identified as fusaric acid (FA) and 9,10‐dehydrofusaric acid (DFA). Both FA and DFA production began in the first week of growth, increasing gradually to their maxima after 4 weeks. The highest level of pathogenic activity of the fungus was obtained after three or more weeks of fungal growth. It can be concluded that in order to produce high levels of toxin and pathogenic activity, the fungus should be grown under illumination without shaking for 4 weeks.     

Solid substrate for production of Alternaria alternata conidia: a potential mycoherbicide for the control of Eichhornia crassipes (water hyacinth)

Eichhornia crassipes (water hyacinth), a major aquatic weed in India, is one of the targets in a biological weed control research programme in India. A fungal pathogen, Alternaria alternata, is being evaluated as a biological control agent for this weed. The feasibility of solid substrate for the mass production of A. alternata has been examined. Conidia production and virulence of A. alternata were affected by temperature, light and incubation period. The highest number of conidia were produced on rice seed followed by wheat, sorghum, maize seeds and cornmeal at 20°C when exposed to near‐ultraviolet than on the other substrates, while the least conidia were observed on these substrates under light conditions. At 20°C, large numbers of virulent conidia were produced on rice seeds after 4 weeks of incubation under constant dark conditions. Henceforth, the use of rice seeds as a solid substrate for production of A. alternata could be a feasible method to produce conidia in a village co‐operative scenario in India.     

Effect of moisture, inoculum production, and planting substrate on disease reaction of field bindweed (Convolvulus arvensis L.) to the fungal pathogen, Phomopsis convolvulus

Phomopsis convolvulus Ormeno is being evaluated as a possible bioherbicide for field bindweed (Convolvulus arvensis L.) control. A granular barley formulation was applied pre-emergence onto the soil surface of pots containing pregerminated field bindweed seeds. Covering the pots with transparent plastic bags immediately after application increased disease incidence and resulted in up to 81% reduction in above-ground dry biomass, whereas a treatment of interrupted dew periods (8 h day^-1) for six days, resulted in only 56% biomass reduction. The size of container used for producing and for incubating the fungus granules had no significant effect on disease incidence and subsequent weed control of field bindweed. Likewise, no significant differences in efficacy were observed using inoculum that was milled once and then sieved or repeatedly milled and non-sieved. For early application dates, the use of two different planting substrates led to major differences in disease development. Pre-emergence application of inoculum on the surface of field collected soil on the same day that field bindweed seeds were planted resulted in an 81% mortality of seedlings emerging. In contrast, only 50% of emerging seedlings were killed when inoculum was applied on the surface of peat moss. Findings in this study indicate that moisture conditions and planting substrate may affect disease incidence and subsequent control of field bindweed by pre-emergence application of the selective fungal pathogen, P. convolvulus.     

稻田稗属杂草致病菌的分离与鉴定

稗草Echinochloa crus-galli作为稻田难以防除的恶性杂草,严重影响水稻产量。为发掘具有防治稻田稗草潜力的生防菌,本研究从江苏、广西、贵州三省采集感病稗草,采用组织分离法获得10株对稗草致病性较强的优势菌株。通过观察优势病原菌的形态特征,结合rDNA-ITS、GAPDH、EF-1α基因片段序列分析确定10株菌分别为新月弯孢Culvularia lunata、尖角突脐蠕孢Exserohilum monoceras、禾长蠕孢菌Setosphaeria rostrata、稻平脐蠕孢Bipolaris oryzae、梭形突脐蠕孢Exserohilum fusiforme、澳大利亚弯孢Curvularia australiensis、双色平脐蠕孢Bipolaris bicolor、高粱附球菌Epicoccum sorghinum、亚隔孢壳属Didymella americana和亚隔孢壳属Didymella pinodella。该研究丰富了稗草病原菌生物资源,为进一步开发稻田生物除草剂提供了新材料。     

Tracing the origins of White Tip disease of Cirsium arvense and its causal agent,Phoma macrostoma

Cirsium arvense (Asteraceae) is known as creeping thistle in its native range in the UK and Canada thistle in its invasive North American range. Recently, the fungus Phoma macrostoma was registered in Canada as a bioherbicide in turfgrass, where it causes severe chlorosis (White Tip disease) and death of C. arvense and other broadleaved weeds. It was hypothesised that the disease originated in the UK on its thistle host and, therefore, that fungal isolates from both countries should be biologically and genetically similar. Twenty‐six strains in the genus Phoma– isolated during surveys in the UK – were compared morphologically with the type culture of P. macrostoma, tested for bioherbicidal activity using the inoculum mat bioassay and genetically screened with bioherbicide‐specific primers. White tip disease was found to be restricted to the eastern and southern counties of England. Phoma macrostoma was isolated consistently from diseased bleached tissues. Bioherbicidal isolates of P. macrostoma occupy a unique clade, which is phylogenetically distinct but morphologically indistinguishable from the type culture. Most isolates from the UK had the same bioherbicidal activity and similar genetic make‐up as strain SRC 94‐44B, the active ingredient in the registered Canadian product. The origin of all bioherbicidal strains found to date has a clear presence in both Canada and the UK, with strong genetic similarities, supporting the view of a common ancestry. Thus, on the evidence presented, the ‘white tip’ clade of P. macrostoma evolved in southern England. Therefore, the bioherbicide based on strain SRC 94‐44B should also be eligible for registration in the UK, based on the pest risk assessment data already available.     

Variability in aggressiveness,cultural characteristics,cercosporin production and fatty acid profile of Cercospora piaropi,a biocontrol agent of water hyacinth

The extent of variation in aggressiveness, growth and pigmentation in culture, phytotoxin production and fatty acid profile were determined in a population of 55 isolates of Cercospora piaropi, a fungus used as a biocontrol agent of the aquatic weed water hyacinth (Eichhornia crassipes). Besides differences in the colour of mycelium and diffusible pigments in culture, isolates of C. piaropi grown under standard conditions differed significantly in their ability to produce the phytotoxin cercosporin, as well as in aggressiveness and growth rate. A positive correlation existed between the ability of the isolates to produce cercosporin and their aggressiveness, and a negative correlation between growth rate and cercosporin production or growth rate and aggressiveness. Based on thin‐layer chromatographic separation of extracts and comparison with beticolin‐1, used as a standard, there was no evidence of production of beticolins. In discriminant analysis, fatty acid methyl ester (FAME) profiles had low resolution for differentiating populations among isolates of the fungus, and the level of resolution was influenced by the age of the colonies. Diffusible pigments in culture and cercosporin production are useful adjuncts to aggressiveness screening for choosing the most effective isolate of C. piaropi for biological control.