Take-all Pathogen Research Articles

Multifunctional manganese-based carboxymethyl chitosan hydrogels for pH-triggered pesticide release and enhanced fungicidal activity

Carboxymethyl chitosan (CMCS) has emerged as a promising biopolymer carrier for controlled release formulations of pesticide. In this study, manganese-based carboxymethyl chitosan hydrogel was facilely prepared to encapsulate and release fungicide prothioconazole in a controllable manner. The loading content (LC) and encapsulation efficiency (EE) of prothioconazole were optimized by orthogonal test. When scaled up under the optimal condition, the corresponding LC and EE were 22.17 % and 68.38 %, respectively. The result showed that the pH-triggered release behavior of prothioconazole for the hydrogels was consistent with swelling behavior. The pesticide rapidly released in neutral and slightly alkaline solutions than in acidic conditions. Moreover, the prepared hydrogel showed enhanced fungicidal ability against wheat take-all pathogen (Gaeumannomyces graminis var. tritic) compared to that of prothioconazole technical material. This research seeks to provide a promising approach to develop metal and polysaccharide-based hydrogels to control the pesticide release and reduce pesticide use in sustainable agriculture application.

Construction of a recombinant strain of Pseudomonas fluorescens producing both phenazine-1-carboxylic acid and cyclic lipopeptide for the biocontrol of take-all disease of wheat

The primary mechanism of biocontrol by Pseudomonas fluorescens strains HC1–07 and HC9–07 is production of a cyclic lipopeptide (CLP) and phenazine-1-carboxylic acid, respectively. We introduced the seven-gene operon for the synthesis of phenazine-1-carboxylic acid (PCA) from P. synxantha 2–79 into Pseudomonas fluorescens HC1–07rif to determine if the biocontrol activity of the recombinant strain HC1–07PHZ increased against Gaeumannomyces graminis var. tritici, causal agent of take-all disease of wheat. In vitro inhibition assays showed that strain HC1–07PHZ consistently inhibited the hyphal growth of three isolates of the take-all pathogen on plates of both PDA and KMB, and the recombinant consistently inhibited G. graminis. var. tritici to a greater extent than the wild type. Strain HC1–07PHZ applied at a dose of 102 CFU seed−1 suppressed take-all better than strains HC1–07rif and HC9–07rif applied either individually or in combination. When the dose of the bacteria was increased to 104 CFU seed−1, the strain combination HC1–07rif + HC9–07rif showed significantly better disease suppression than did HC1–07rif, HC9–07rif or HC1–07PHZ applied individually. However, when the bacterial dose was increased to 107 CFU seed−1, strains HC1–07rif and HC1–07PHZ showed significantly better disease suppression than HC9–07rif and the combination HC1–07rif + HC9–07rif.

Erratum: Pseudomonas spp. diversity is negatively associated with suppression of the wheat take-all pathogen.

Scientific Reports 6: Article number: 29905; published online: 23 August 2016; updated: 10 October 2016.

Pseudomonas spp. diversity is negatively associated with suppression of the wheat take-all pathogen.

Biodiversity and ecosystem functioning research typically shows positive diversity- productivity relationships. However, local increases in species richness can increase competition within trophic levels, reducing the efficacy of intertrophic level population control. Pseudomonas spp. are a dominant group of soil bacteria that play key roles in plant growth promotion and control of crop fungal pathogens. Here we show that Pseudomonas spp. richness is positively correlated with take-all disease in wheat and with yield losses of ~3 t/ha in the field. We modeled the interactions between Pseudomonas and the take-all pathogen in abstract experimental microcosms, and show that increased bacterial genotypic richness escalates bacterial antagonism and decreases the ability of the bacterial community to inhibit growth of the take-all pathogen. Future work is required to determine the generality of these negative biodiversity effects on different media and directly at infection zones on root surfaces. However, the increase in competition between bacteria at high genotypic richness and the potential loss of fungal biocontrol activity highlights an important mechanism to explain the negative Pseudomonas diversity-wheat yield relationship we observed in the field. Together our results suggest that the effect of biodiversity on ecosystem functioning can depend on both the function and trophic level of interest.

Generic names in Magnaporthales

The order Magnaporthales comprises about 200 species and includes the economically and scientifically important rice blast fungus andthe take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthales with their type species and available gene and genome resources. The polyphyletic Magnaporthe 1972 is proposed for suppression, and Pyricularia 1880 and Nakataea 1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

Isolation and identification of Bacillus subtilis strain YB-05 and its antifungal substances showing antagonism against Gaeumannomyces graminis var. tritici

In this study, 98 putative Bacillus strains were isolated from wheat rhizospheric soil. Among the isolated strains, six showed strong inhibitory effects against the wheat take-all pathogen, Gaeumannomyces graminis var. tritici. One of the strains that showed significant inhibitory activity, YB-05, was identified as Bacillus subtilis based on a phylogenetic analysis of its 16S rDNA gene sequence, the results of the PCR analysis and cloning of its antifungal genes, its morphological characteristics and its physiological and biochemical properties. When tested with a dual-culture, cup–disc method and laboratory greenhouse studies, strain YB-05 was found to be superior to chemical treatment for control of the plant pathogen G. graminis var. tritici. After liquid culture, various antimicrobial substances in the culture medium were detected by high-performance liquid chromatography and high-resolution mass spectrometry, and the existence of their corresponding genes was verified by PCR analysis.

Soil microbes are linked to the allelopathic potential of different wheat genotypes

Soil micro-circumstance and biological stress resistance were studied to validate our hypothesis that the allelopathic potential that was enhanced by breeding resulted partially from rhizophere microbes associated with the different varieties. The rhizosphere soils from four wheat genotypes with different allelopathic potential were collected so as to compare their soil micro-environments and bio-pressure tolerances. The levels of these three categories such as bacteria, fungi, and actinomycetes ranged among 1.54–26.59 × 106, 0.43–4.12 × 104, and 1.36–18.25 × 105 CFU/g soil, respectively. Wheat 22 Xiaoyan with greater allelopathic potential had higher levels of microorganisms than the other three genotypes having weak allelopathy. The soil microbial carbon and nitrogen analyses suggested that wheat could create an active microhabitat with high activities of key soil enzymes such as urease, catalase, sucrase, and dehydrogenase. Using the approximate concentrations detected in wheat rhizosphere soils, the leachates of all four wheat materials significantly inhibited the growth of the weed Descurainia sophia and take-all pathogen Gaeumannomyces graminis var. Tritici. Wheat exudates provided carbon and nitrogen resources for the relevant microorganism. Meanwhile, the rhizosphere soil microbes contributed to allelopathic potential of wheat by positive feedback.

AN IN-VITRO SCREENING METHOD TO STUDY THE ACTIVITY POTENTIAL OF BIOFERTILIZERS BASED ON TRICHODERMA AND BACILLUS SP.

Little scientific information on efficiency of different commercial biofertilizers restricts setup of further reproducible pot or field experiments and hence, provides lack of evidence of biofertilizer application in plant growth promotion and disease suppression. In the present experiment, efficiency of four commercial Trichoderma and one Bacillus biofertilizer was screened by a bio-indicator plant, cucumber (Cucumis sativus L.) under controlled laboratory conditions. Inoculation of cucumber seeds with different commercial biofertilizers significantly increased the germination rates (ca. 20–25%) and stimulated other growth parameters. In seedling establishment test, biofertilizers inoculated cucumber seedlings showed significant higher root dry weight (ca. 32 to 96%), leaf area (ca. 60 to 140%), root length (ca. 30%) and shoot dry weight (ca. 88%) in two weeks culture period compare to that of the control. Additionally, in-vitro antagonistic activity against take-all pathogen (Gaeumannomyces graminis var. tritici, Ggt) in wheat and phosphate solubilizing activity was demonstrated for Trichoderma biofertilizer. However, in-vitro solubilization of Mn was not detected. The results suggested that the potential activity of different commercial biofertilizers could be easily screened within several days with the described rapid bio-test by increasing seed germination, and improving growth and growth related parameters of cucumber grown in nutrient solution under controlled culture system.

Take-all of Wheat and Natural Disease Suppression: A Review.

In agro-ecosystems worldwide, some of the most important and devastating diseases are caused by soil-borne necrotrophic fungal pathogens, against which crop plants generally lack genetic resistance. However, plants have evolved approaches to protect themselves against pathogens by stimulating and supporting specific groups of beneficial microorganisms that have the ability to protect either by direct inhibition of the pathogen or by inducing resistance mechanisms in the plant. One of the best examples of protection of plant roots by antagonistic microbes occurs in soils that are suppressive to take-all disease of wheat. Take-all, caused by Gaeumannomyces graminis var. tritici, is the most economically important root disease of wheat worldwide. Take-all decline (TAD) is the spontaneous decline in incidence and severity of disease after a severe outbreak of take-all during continuous wheat or barley monoculture. TAD occurs worldwide, and in the United States and The Netherlands it results from a build-up of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. during wheat monoculture. The antibiotic 2,4-DAPG has a broad spectrum of activity and is especially active against the take-all pathogen. Based on genotype analysis by repetitive sequence-based-PCR analysis and restriction fragment length polymorphism of phlD, a key 2,4-DAPG biosynthesis gene, at least 22 genotypes of 2,4-DAPG producing fluorescent Pseudomonas spp. have been described worldwide. In this review, we provide an overview of G. graminis var. tritici, the take-all disease, Pseudomonas biocontrol agents, and mechanism of disease suppression.

Purification and characterization of a novel extracellular β-1,3-glucanase complex (GluGgt) secreted by Gaeumannomyces graminis var. tritici

β-1,3-Glucanases produced by the take-all disease pathogen Gaeumannomyces graminis var. tritici (Ggt) have been suggested to be implicated in infection and colonization process of the pathogen in wheat. For further studying the role of these enzymes in pathogenesis by cytochemical technique, an extracellular β-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH4)2SO4 precipitation, hydrophobic-interaction, anion-exchange and size-exclusion chromatography. The complex consisted of two interconvertible isoforms (Ia and Ib), both active proteins Ia and Ib appeared to be glycosylated in native polyacrylamide gel electrophoresis (PAGE). The pI of the active proteins Ia and Ib determined by IEF-PAGE were 6.3 and 6.4, respectively. GluGgt yielded two subunits with molecular masses of 66.2 and 56.0 kDa, respectively, in 15% SDS–PAGE. The complex had a pH optimum of 5.0 and the optimal temperature was 50°C. The enzyme complex proved to be stable at a temperature up to 60°C and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mn2+ ions and moderately inhibited in the presence of Hg2+ and Co2+ ions and KMnO4. The Km of GluGgt was estimated to be 1.08 mg ml−1 for laminarin as substrate and the Vmax 0.20 μmol min−1.

Diversity, Virulence, and 2,4-Diacetylphloroglucinol Sensitivity of Gaeumannomyces graminis var. tritici Isolates from Washington State

We determined whether isolates of the take-all pathogen Gaeumannomyces graminis var. tritici become less sensitive to 2,4-diacetylphloroglucinol (2,4-DAPG) during wheat monoculture as a result of exposure to the antibiotic over multiple growing seasons. Isolates of G. graminis var. tritici were baited from roots of native grasses collected from noncropped fields and from roots of wheat from fields with different cropping histories near Lind, Ritzville, Pullman, and Almota, WA. Isolates were characterized by using morphological traits, G. graminis variety-specific polymerase chain reaction and pathogenicity tests. The sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was determined by measuring radial growth of each isolate. The 90% effective dose value was 3.1 to 4.4 microg ml(-1) for 2,4-DAPG-sensitive isolates, 4.5 to 6.1 microg ml(-1) for moderately sensitive isolates, and 6.2 to 11.1 microg ml(-1) for less sensitive isolates. Sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was normally distributed in all fields and was not correlated with geographic origin or cropping history of the field. There was no correlation between virulence on wheat and geographical origin, or virulence and sensitivity to 2,4-DAPG. These results indicate that G. graminis var. tritici does not become less sensitive to 2,4-DAPG during extended wheat monoculture.

Biocontrol evaluation of wheat take-all disease by Trichoderma screened isolates

Wheat take-all disease, caused by Gaeumannomyces graminis var tritici (Ggt), has been observed in different areas of Iran in recent years. Current biocontrol studies have confirmed the effectiveness of the Trichoderma species against many fungal phytopathogens. In this study, biocontrol effects of Trichoderma isolates were evaluated against take-all pathogen. Five (Trichoderma virens T65, Trichoderma virens T90, Trichoderma virens T96, Trichoderma virens T122 and Trichoderma koningii T77) isolates were selected after screening tests for antifungal effects against take-all pathogen and three (Trichoderma koningiopsis T149, Trichoderma brevicompactum T146 and Trichoderma viridecens T150) isolates were used as random selection. Also, Trichodermin B and Subtilin as commercial bioproducts were also used to evaluate biocontrol effects against take-all in greenhouse. In dual culture tests, five (T65, T90, T96, T122 and T77) isolates covered and colonized the colony of the pathogen. Microscopic studies revealed hyphal coiling (hyperparasitism) of isolates T65, T96 and T90 around Ggt hyphae. Culture filtrates and volatile metabolites of all isolates reduced the mycelial growth of take-all pathogen. T96, T65, mixture of eight isolates and Trichodermin B were more effective than other treatments to control the disease and reduced 25 to 55% disease severity and increased 27 to 59% dry weight of shoots and 23 to 58% dry weight of roots compared with control while disease reduction of seed coating method was more than soil treatment application. Although, based on shoots and roots dry weight, all of treatments showed significant differences with healthy plants.

Influence of Pseudomonas fluorescens and arbuscular mycorrhiza on the growth, yield, quality and resistance of wheat infected with Gaeumannomyces graminis

The interaction between the arbuscular mycorrhizal fungus (AMF) Glomus mossea, the plant growth promoting rhizobacteria (PGPR) Pseudomonas fluorescens strain RA56 and the take-all pathogen Gaeumannomyces graminis, were investigated on spring wheat (Triticum aestivum L.). The subsequent effects on growth, yield, seed quality and resistance of plants were investigated under greenhouse conditions over a 2-year period. Plants inoculated with G. mosseae or P. fluorescens strain RA56 had a lower incidence of the root take all disease than untreated ones. The combined inoculation of P. fluorescens strain RA56 and AMF showed the best results in reducing pathogen impact at 4 weeks. Furthermore, prior treatment with AMF resulted in an increase in shoot drymass and seed yield in G. graminis infected wheat. The best results in terms of mycorrhiza colonization was recorded with 10% of mycorrhiza inoculum.

Role of 2,4‐Diacetylphloroglucinol‐Producing Fluorescent Pseudomonas spp. in the Defense of Plant Roots

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.

Effect of Organic Management of Soils on Suppressiveness to Gaeumannomyces graminis var. tritici and its Antagonist, Pseudomonas fluorescens

Organic management of soils is generally considered to reduce the incidence and severity of plant diseases caused by soil-borne pathogens. In this study, take-all severity on roots of barley and wheat, caused by Gaeumannomyces graminis var. tritici, was significantly lower in organically-managed than in conventionally-managed soils. This effect was more pronounced on roots of barley and wheat plants grown in a sandy soil compared to a loamy organically-managed soil. Fluorescent Pseudomonas spp. and in particular phlD+ pseudomonads, key factors in the take-all decline phenomenon, were represented at lower population densities in organically-managed soils compared to conventionally-managed soils. Furthermore, organic management adversely affected the initial establishment of introduced phlD+ P. fluorescens strain Pf32-gfp, but not its survival. In spite of its equal survival rate in organically- and conventionally-managed soils, the efficacy of biocontrol of take-all disease by introduced strain Pf32-gfp was significantly stronger in conventionally-managed soils than in organically-managed soils. Collectively, these results suggest that phlD+ Pseudomonas spp. do not play a critical role in the take-all suppressiveness of the soils included in this study. Consequently, the role of more general mechanisms involved in take-all suppressiveness in the organically-managed soils was investigated. The higher microbial activity found in the organically-managed sandy soil combined with the significantly lower take-all severity suggest that microbial activity plays, at least in part, a role in the take-all suppressiveness in the organically-managed sandy soil. The significantly different bacterial composition, determined by DGGE analysis, in organically-managed sandy soils compared to the conventionally-managed sandy soils, point to a possible additional role of specific bacterial genera that limit the growth or activity of the take-all pathogen.

Changes in populations of rhizosphere bacteria associated with take-all disease of wheat.

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant Pseudomonas spp. and a phlD(+) Pseudomonas sp. showed any significant ability to inhibit G. graminis var. tritici directly. When cultured strains were mixed with the inhibitory phlD(+) Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD(+) pseudomonads.

A new growth medium for the study of siderophore-mediated interactions

A microbial growth medium, RSM, was developed to study the role of siderophores (microbial Fe-transport compounds) in the inhibition of the take-all pathogen, Gaeumannomyces graminis var. tritici, by Pseudomonas putida strain B10. The inorganic constituents of the medium were designed to mimic the rhizosphere while the organic composition was designed to promote rapid growth and siderophore production. The antibiosis experiments were highly reproducible and the antagonism appeared to be due to production of pseudobactin, the siderophore of B10. On plates amended with chrome azurol S, G. graminis did not produce siderophores while other fungi did. The growth of G. graminis on plates prepared with Fe chelate buffers was inhibited at a free ferric ion concentration of 10−24.6 M, although three other fungi were not inhibited, even at 10−25.5 M, presumably due to their greater production of siderophores. In liquid medium amended with Fe chelate buffers, both the doubling time and the lag phase of P. putida increased as the free ferric ion concentration was reduced. A wide variety of fungi and bacteria were found to grow on this medium. Because the inorganic composition of RSM is based on that of the rhizosphere, the development of this medium may be a first step towards the study of the chemistry and biology of the rhizosphere under well defined conditions.

Influence of weight of seed on hyphal growth of the take-all pathogen, Gaeumannomyces graminis var. tritici, in wheat roots grown in sand culture

This study examined extent of hyphal growth of Gaeumannomyces graminis var. tritici (Ggt) in seminal roots of wheat seedlings grown from seed of varying weight (12-60 mg). Hyphal invasion, measured microscopically in the inner cortex, endodermis and stele of root sections and in the early formation of stelar lesions, was found to be influenced in a complex manner by weight of seed. As weight of seed was increased, invasion of the inner cortex decreased linearly while invasion of stelar tissue varied in a non linear manner. These findings show that weight of seed must be considered when comparing levels of infection in seedlings of different cultivars of wheat.

Effect of Certain Isolates of Soil Fungi on Take-All of Wheat

Of 41 soil fungi isolated from the roots of wheat 17 reduced the severity of take-all of wheat caused by Gaeomannomyces graminis var. tritici in unsterile yellow sand. Some isolates showing potential as biocontrol agents were tested in unsterile wheat-field soil. In such tests Phialophora fastigiata, P. malorum, P. hoffmanniiM and Penicillium patulam gave significant protection against take-all, but the best control was given by three species of Fusarium. The plants exposed to F. oxysporum and the take-all pathogen together produced shoots significantly more vigorous than uninoculated controls receiving no fungi. F. oxysporum also significantly increased the shoot weight of wheat in the absence of the pathogen.