Physiological characterization of the dandelion bioherbicide,Sclerotinia minor IMI 344141

The fungus Sclerotinia minor (IMI 344141) is being developed as a biological control for dandelion and other broadleaf weeds in turfgrass environments. Being a microbial pest control agent (MPCA), the S. minor strain must be characterized to show relatedness to like organisms and to distinguish the MPCA from related microorganisms. Phenotypic variation among 30 isolates of S. minor, collected from different regions and hosts, was studied on potato dextrose agar (PDA) and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates. There was high diversity (0.6) among the mycelial compatibility groups (MCG) as seven multi-member and 11 single member groups were recognized. Isolates were categorized into highly virulent, virulent, moderately virulent, and hypo virulent based on 48 h post mycelial growth on detached dandelion leaves. When assessed on dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest in biocontrol efficacy, reduction of above- and below-ground biomass, and reduction in dandelion survival. Oxalic acid production was not correlated with isolate aggressiveness or growth rate and did not vary among isolates of the same MCG. IMI 344141 can be phenotypically distinguished from the other tested S. minor isolates by performing vegetative compatibility testing and counting sclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 produces <100 sclerotia/plate.     

Effect of plant age and turfgrass competition on the efficacy of the Sclerotinia minor granular bioherbicide on broadleaf plantain and prostrate knotweed

Broadleaf plantain and prostrate knotweed are important weeds of turfgrass systems. The fungus Sclerotinia minor Jagger (IMI 344141) has been registered as a biological herbicide (Sarritor?) for dandelion (Taraxacum officinale) in Canadian turfgrass habitats. The objective of this study was to evaluate the effect of plant age and turfgrass environment on the efficacy of S. minor against two additional weeds; broadleaf plantain and prostrate knotweed. The turfgrass environment alone exerted significant reduction of above and below ground biomass of broadleaf plantain, to the same magnitude as the S. minor treatment in a grass-free environment. Prostrate knotweed biomass, however, was not reduced to this extent by competition with turfgrass. In the presence of grass, S. minor caused a significant biocontrol effect on all studied variables with more than 90% above ground damage on both weed species. Severe damage occurred on 3–6-week-old plantains with 100% above and below ground reduction, although smaller dry weight reductions were observed on older plantains. Treatment with S. minor reduced the dry matter of 3–5-week-old prostrate knotweed by 65–85%, but less damage occurred on older prostrate knotweed. The bioherbicide fungus is destructive for both species, but variation in area of contact due to different growth forms, growth rates and resource allocation patterns due to different life forms resulted in different biocontrol efficacy on the two species. Control of broadleaf plantain was effective – similar to that previously reported for dandelion – but control of prostrate knotweed was only partial.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Impact of mowing and weed control on broadleaf weed population dynamics in turf

Abstract

Various turf management activities may influence weed population dynamics and interfere with weed control. The effects of a biocontrol agent, Sclerotinia minor, a chemical herbicide, Killex?, and mowing height on broadleaf weed dynamics were examined in two turfgrass stands for two consecutive years. Mowing did not reduce the population densities of dandelion or the ground cover of broadleaf weeds. In the second year, mowing significantly reduced white clover density, but significantly increased broadleaf plantain density, particularly at the closest mowing height (3–5 cm). Apart from the close height, the S. minor and Killex? treatments were equally effective in suppression of dandelion, white clover, broadleaf plantain, and prostrate knotweed in the second year. Common mallow increased in the herbicide treated plots and other species including yellow woodsorrel, yellow toadflax and lambsquarters increased in abundance in plots mowed at the 3–5 cm height and in plots treated with Killex?. Significant differences between the Killex? and S. minor treatments on dandelion population dynamics were rarely present and did not favour either treatment. S. minor did not damage the turfgrass, but Killex? reduced turf quality in 25% of the plots. The application of S. minor with a regular, medium height (～7 cm) mowing regime was highly effective in controlling broadleaf weeds in temperate Kentucky bluegrass turf.     

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent

Aims: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. Methods and Results: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot‐grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. Conclusions: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil‐borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. Significance and Impact of the Study: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.     

In vitro identification ofTrichoderma harzianum as a potential antagonist of plant pathogens

When compared with a range of colonists of straw and other potential antagonists of glasshouse and cereal pathogens,Trichoderma harzianum IMI 275950 exhibited the greatest biocontrol activity in vitro. This activity was also observed in a gnotobiotic lettuce bioassay against the pathogenSclerotinia sclerotiorum. Activity was dependent on antagonist/pathogen inoculum ratio and temperature.     

Bacillus altitudinis strain AMCC 101304: a novel potential biocontrol agent for potato common scab

ABSTRACT

Potato common scab, caused by Streptomyces spp., is one of the leading causes of heavy commercial losses in the potato industry and is thus one of the most serious plant diseases worldwide. This study identified and assessed potential biocontrol agents against potato common scab. In total, 110 isolates were obtained through antagonistic tests; among which, Bacillus sp. strain AMCC 101304 was found to be most effective at inhibiting the potato common scab pathogen, Streptomyces scabies. Bacillus sp. strain AMCC 101304 was finally identified as Bacillus altitudinis by morphological observation, physiological and biochemical experimentation, as well as 16S rRNA sequencing and phylogenetic analysis. Pot experiments were conducted twice (in spring and autumn) to verify the biocontrol effect of B. altitudinis AMCC 101304 against potato common scab. In spring, the control efficiency reached 76.34%. In autumn, the disease incidence was reduced from 100% to 34.19% (one treatment with strain AMCC 101304) and 38.42% (two treatments with strain AMCC 101304), and the control efficiency reached 82.50% (one application) and 78.43% (two applications). The present study demonstrated the potential of an isolate, identified as B. altitudinis AMCC 101304, as an effective biocontrol agent for future use in the field.     

Novel strains of Bacillus,isolated from compost and compost-amended soils,as biological control agents against soil-borne phytopathogenic fungi

A stepwise screening strategy made it possible to identify five new Bacillus spp. strains for biocontrol of Rhizoctonia solani, Sclerotinia minor and Fusarium solani. In vitro and in vivo biocontrol activity and M13-PCR DNA-fingerprinting led to the selection of these valuable biological control agents (BCAs) from a wide collection of over 250 candidates. At the end of this selection, the highest potential antagonists were identified at species level by 16S-rRNA gene sequence analysis, and results assigned them to Bacillus subtilis group as Bacillus amyloliquefaciens- and Bacillus methylotrophicus-related strains. In the current study, spore-forming bacteria provided substantial biocontrol of telluric diseases on cress and other different host plants. The strains named 15S and 09C were effective in disease control on Brassica oleracea/R. solani pathosystem, whereas Sclerotinia drop of lettuce was reduced by treatments with the strains 17S and 08C. Finally, the strains 17S and 12S were equally effective to control potato Fusarium rot. The evident zone of inhibition seen in dual culture plates suggested antibiosis-like antagonisms as the main mechanisms used by these bacterial isolates in interaction with the pathogens. Additionally, the API-ZYM method revealed constitutive activity of certain extracellular enzymes that could be involved in plant fortification. Bacillus strains isolated from compost and compost-amended soils are promising BCAs that have potential for practical application as biofungicides.     

Homothallism in Sclerotinia minor

Sclerotinia species are sexually reproducing ascomycetes. In the past S. minor and S. sclerotiorum, have been assumed to be homothallic because of the self-fertility of colonies derived from single ascospores. S. trifoliorum has previously been shown to be bipolar heterothallic due to the presence of four self-fertile and four self-sterile ascospores within a single ascus [Uhm, J.Y., Fujii, H., 1983a. Ascospore dimorphism in Sclerotinia trifoliorum and cultural characters of strains from different-sized spores. Phytopathology 73: 565–569]. However, isolates of S. minor and S. sclerotiorum were proven to be homothallic ascomycetes, by self-fertility of all eight ascospores within an ascus. Apothecia were raised from all eight ascospores of a single tetrad from four isolates of S. minor and from an isolate of S. sclerotiorum, indicating that inbreeding may be the predominant breeding mechanism of S. minor. Ascospores from asci of S. minor and S. sclerotiorum were predominantly monomorphic, but rare examples of ascospore dimorphism similar to S. trifoliorum were found.     

The use of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> P13 to control sclerotinia stem rot (<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>) of oilseed rape

Sclerotinia stem rot (SSR) caused by the fungus Sclerotinia sclerotiorum has been an increasing threat to oilseed rape (Brassica napus L.) cultivation. Efficient and environment—friendly treatments are much needed. Here we focus on microbial control. The Pseudomonas fluorescens P13 that was isolated from oilseed rape cultivation soil, proved to be a useful biocontrol strain for application. Morphology, physiological and biochemical tests and 16S rDNA analysis demonstrated that it was P. fluorescens P13 and that it had a broad antagonistic spectrum, significantly lessening the mycelial growth of S. sclerotiorum by 84.4% and suppressing sclerotial formation by 95–100%. Scanning electron microscopy studies attested that P13 deformed S. sclerotiorum mycelia when they were cultured together. P13 did not produce chitinase but did produce hydrogen cyanide (HCN) which was likely one of the antagonistic mechanisms. The density of P13 remained at a high level (≥10⁶ CFU/ml) during 5 weeks in the rhizosphere soil and roots. P13 reduced SSR severity at least by 59% in field studies and also promoted seedling growth (p<0.05) at the seedling stage. From these data, our work provided evidence that P13 could be a good alternative biological resource for biocontrol of S. sclerotiorum.     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

Effets de Traitements Fongicides aux Imides Cycliques sur les Populations de Sclerotinia minor dans le Sol

About the effects of cyclic imides fungicidal treatments on soil populations of Sclerotinia minor When S minor selerotia were incubated in natural soils, they were gradually attacked by a varied antagonistic flora. This phenomenon was seriously slackened when cyclic imides (iprodione of vinclozolin) at 25 or 50 mg.l^-1 were incorporated into the soil, in laboratory trials or in normal conditions of cultivation. These fungicides (which are currently used to control S. minor) seemed to protect selerotia by preventing the development of antagonistic fungi. Selerotial germination was inhibited by vinclozolin but, if the duration of contact was less than 5 weeks, the phenomenon was fully reversible, even in the presence of very high concentrations (500 mg.l^-1). When an inoculum of Trichoderma sp. was added to natural soils, selerotia were rapidly damaged. An even faster decrease of S. minor populations resulted from the incorporation of cyclic imides into these Trichoderma-enriched soils. These organisms are not very susceptible to such fungicides. It is suggested that a joint utilization of these biological auxiliaries and of cyclic imides should be a way to avoid the appearance of a biological unbalance due to an exclusive use of fungicides.     

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan® PCR

Aims: To develop a strain‐specific TaqMan^® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan^® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan^® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.     

Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7

In the present study, the endophytic bacterium Bacillus subtilis strain Em7 (GU258545.1) was evaluated as a biological control agent for Sclerotinia sclerotiorum on oilseed rape. In petri dish, strain Em7 not only strongly inhibited pathogen mycelium growth but also germination of sclerotia at concentrations between 10⁹ and 10¹¹ colony forming unit (CFU)·ml^?1. Scanning electron microscopy and transmission electron microscopy studies revealed that in the presence of strain Em7, hyphae of S. sclerotiorum showed leakage and disintegration of hyphal cytoplasm. Furthermore, the strain Em7 showed a broad antifungal spectrum on mycelium growth of numerous important plant pathogenic fungi. Light microscopic observations revealed that strain Em7 caused morphological alterations including increased branching, swelling and collapse of cytoplasm. In the greenhouse, spray treatments of cell suspensions of strain Em7 (1×10⁹ CFU·ml^?1) reduced leaf and stem rot incidence and severity in the seedling and blossom stage. The control efficacy was higher when strain Em7 cell suspension was applied one day prior to inoculation of the pathogen than after inoculation. Three-year field trials showed that two applications of strain Em7 cell suspension at blossom stage significantly reduced disease incidence and severity by 50–70%. There was no significant difference in control efficacy among treatments with strain Em7 cell suspension and the fungicides containing carbendazim or tebuconazole (P = 0.05). Thus, our results strongly suggest that B. subtilis strain Em7 is a promising biological control agent for control of oilseed rape Sclerotinia stem rot.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Efficient transformation and expression of the glucanase gene from Bacillus megaterium in the biocontrol strain Streptomyces lydicus A02

Streptomyces have been used extensively as the biocontrol agents due to their ability to produce various antimicrobial compounds, such as antibiotics and hydrolytic enzymes. Streptomyces lydicus strain A02, which was isolated from the soil of suburban forest field in Beijing (China), is capable of producing natamycin and has proved to be a potential biocontrol agent to several plant fungal diseases, including wilts caused by Fusarium oxysporum f. spp. However, hydrolytic enzymes like glucanase have not been detected in S. lydicus A02 on CMC-Na plates by congo red staining. Glucanase, a pathogenesis-related (PR) protein, degrades fungal cell walls and has been widely used as antifungal agent in plant protection. Therefore, a recombinant S. lydicus expressing a glucanase gene, which was cloned from the biocontrol strain Bacillus megaterium L103 and driven by the Streptomyces erythraea ermE* promoter, was constructed in this study. The engineered S. lydicus AG02 shared a similar yield of natamycin with the wild-type A02 strain. Compared to the wild-type strain A02, the engineered S. lydicus AG02 had a remarkably higher glucanase activity, as well as antifungal activity to F. oxysporum f. sp. conglutinans, F. oxysporum f. sp. niveum and Rhizoctonia cerealis. This demonstrated the improved biocontrol effect of S. lydicus AG02 attributed to transforming the exogenous glucanase from B. megaterium, which acted synergistically with natamycin to increase the antifungal activity of the strain.     

Expressed sequence tags-based identification of genes in a biocontrol strain Trichoderma asperellum

Trichoderma asperellum, a filamentous soil fungus, is an effective biocontrol agent against many fungal plant pathogenic species. In the present study, we investigated the biological control properties of the strain T. asperellum T4. T. asperellum fermentation products significantly decreased the ability of Rhizoctonia solani and Sclerotinia sclerotiorum to infect rice and soybean, respectively. To further elucidate the biocontrol mechanisms of T. asperellum at the molecular level, a cDNA library was constructed from its mycelium. In total, 3114 expressed sequence tags (ESTs) were generated, which represented 1,554 unigenes, including 354 contigs and 1,200 singletons. Among these unigenes, 731 represented known genes while 823 were novel genes. Forty-six unigenes potentially involved in biocontrol processes were identified from the EST collection. Among them, the expressions of 16 genes were studied, and 15 genes were highly differentially regulated during confrontation with 2 phytopathogens, suggesting that they play roles in the T. asperellum response to phytopathogens. Our study may provide helpful insight in the mechanism of biocontrol by T. asperellum T4 against plant pathogens.     

Persistence of the nematophagous fungus Paecilomyces lilacinus strain 251 in soil under controlled conditions

The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study.     

八角炭疽病拮抗放线菌RX2-2的分离鉴定及其生物活性评价

[背景]八角炭疽病主要是由哈锐炭疽菌(Colletotrichum horii)引起的真菌性病害,给八角产业带来严重的经济损失.[目的]从八角根系土壤中分离筛选对哈锐炭疽菌具有拮抗作用的放线菌菌株,并对其进行种属鉴定及抗菌活性评价.[方法]采用稀释涂布平板法分离放线菌,并以哈锐炭疽菌作为指示菌,利用平板对峙法筛选具有高...     

Introduced Pseudomonas fluorescens VUPf5 as an important biocontrol agent for controlling Gaeumannomyces graminis var. tritici the causal agent of take-all disease in wheat

About 900 bacterial strains obtained from plants rhizosphere from different areas of Iran and biocontrol ability were widely survey in vitro and greenhouse conditions against Gaeumannomyces graminis var. tritici the causal agent of take-all disease. Finally the best strain (VUPf5 strain) in the additional tests biocontrol ability in vitro and greenhouse conditions was selected for next studies. VUPf5 suppressed of take-all disease 85%. Based on biochemical and morphological tests, this isolate is belonging to the species Pseudomonas fluorescens. This isolate significantly produced the secondary metabolites such as siderophores, hydrogen cyanide, protease, phenazine and volatile metabolites.