Characterisation of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flag smut

To explore effective and ecofriendly means of controlling wheat flag smut (WFS) using biocontrol agents, three endophytic strains (58-2-1, 37-1 and YC-1) of Bacillus sp. were isolated from winter wheat plants in China and identified as Bacillus thuringiensis based on their 16S rDNA sequences as well as phenotypic characteristics. Four morphological (leaf length, root length, dry weight and tiller numbers) and one physiological [root vigour (RV)] parameters of wheat plants treated by strains 58-2-1 and 37-1 were significantly enhanced compared to the control. The soluble sugar contents in the roots of wheat samples treated by the two strains were significantly lower than the control. The resistance of wheat varieties to WFS was investigated by inoculation tests. Of the 12 wheat varieties tested, 6 (Yunhan-618, Bainongaikang-58, Kaimai-20, Zhengmai-9023, 04-zhong-36 and Yanzhan-4110) were identified as WFS-highly resistant (HR), 3 (Pumai-9, Jinboshi-1 and Yunong-202) WFS-moderately resistant (MR), 1 (Yubao-1) WFS-susceptible (S) and 2 (Yumai-012 and Yunong-416) WFS-highly susceptible (HS) varieties. The Urocystis tritici-induced yield loss on the S/HS varieties was significantly higher than that on the HR/MR ones. The strains 58-2-1 and 37-1 had control efficacies of 6.7–100% (av.54.8%) and 33.3–100% (av. 66.5%) on 9 and 7 out of 12 varieties, respectively. The strains 58-2-1 and 37-1 had enhanced yields of 10.2–54.9% (av. 32.9%) and 2.8–43.4% (av. 24.8%) on 10 and 8 out of 12 varieties, respectively. This is the first report on endophytic B. thuringiensis strains isolated from wheat plants with the abilities to suppress WFS and to enhance yields on multiple wheat varieties.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Screening of ESTs from Septoria musiva (teleomorph Mycosphaerella populorum) for detection of SSR and PCR–RFLP markers

The development of an expressed sequence tag (EST) collection for the poplar pathogen Septoria musiva presented an opportunity for the development of locus‐specific polymorphic markers. Five microsatellites simple sequence repeat (SSR) and six polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) markers were identified and tested on three S. musiva populations collected in the north‐central and northeastern regions of North America. The results obtained support previously published random amplified polymorphic DNA (RAPD) data.     

Comparing single- vs. mixed-genotype infections of Mycosphaerella graminicola on wheat: effects on pathogen virulence and host tolerance

Mixed-genotype infections (infections of a host by more than one pathogen genotype) are common in plant-pathogen systems. However their impact on the course of the infection and especially on pathogen virulence and host response to infection is poorly understood. We investigated the effects of mixed-genotype infections on several parameters: host resistance and tolerance, as well as pathogen aggressiveness and virulence. For these purposes, we inoculated three wheat lines with three Mycosphaerella graminicola genotypes, alone or in mixtures, in a greenhouse experiment. For some of the mixtures, disease severity and virulence were lower than expected from infection by the same genotypes alone, suggesting that competition between genotypes was reducing their aggressiveness and virulence. One host line was fully resistant, but there were differences in resistance in the other lines. The two host lines that became infected differed slightly in tolerance, but mixed-genotype infections had no effect on host tolerance.     

Variation in Aggressiveness of Stagonospora nodorum Isolates in North Dakota

Stagonospora nodorum blotch (SNB), caused by Stagonospora nodorum, is an important disease in the northern Great Plains of the United States and in other wheat‐producing regions in the world. SNB can be managed by different strategies including the use of resistant cultivars. Genetic variation in the pathogen populations is one of the important factors in the development of durable resistant cultivars. Our main objective was to determine variation in aggressiveness/virulence in the 40 isolates of S. nodorum collected from various locations in North Dakota. To achieve this goal, we tested the isolates on two susceptible wheat cultivars (cvs ‘ND495’ and ‘Alsen’) and two resistant wheat cultivars (cvs ‘Erik’ and ‘Salamouni’) – two‐leaf‐stage seedlings under controlled conditions. Aggressiveness of each isolate was characterized by the two epidemiological parameters: percent necrotic leaf area (% NLA) and lesion type (LT) 8 days post‐inoculation. The isolates differed significantly (P ≤ 0.05) for % NLA and LT, and were grouped into three aggressiveness groups (AG): low, medium and highly aggressive. Four isolates (S50, S57, S66 and S89) induced 18–26% NLA and were included into the low aggressive group (AG 1). Three isolates (S15, S39 and S89) induced 57–59% NLA and were considered highly aggressive (AG 3). Thirty‐three isolates were medium aggressive (AG 2). No relationship between AG and mating types was observed. There were significant (P ≤ 0.05) differences in % NLA and LT among wheat cultivars. Significant wheat cultivars by isolates interaction was also demonstrated, suggesting evidence for the existence of host specificity in this system. Overall, our results indicate that S. nodorum isolates prevalent in North Dakota varied greatly in their aggressiveness and that AG 3 isolates can be utilized in breeding wheat for resistance to SNB.     

Cloning and Expression Analysis of a Putative ABC Transporter Gene BgABC1 from the Biotrophic Pathogenic Fungus Blumeria graminis f. sp. tritici

A putative ATP‐binding cassette (ABC) transporter gene (BgABC1) was isolated from the biotrophic pathogenic fungus Blumeria graminis f. sp. tritici (Bgt). Analysis of the deduced amino acid sequence of BgABC1 showed that the BgABC1 protein had a conserved nucleotide‐binding fold in the N‐terminus, and six transmembrane domains (TMDs) in the C‐terminus. Analysis of the BgABC1 expression using real‐time polymerase chain reaction showed that expression of the gene was increased significantly when the fungus was growing in wheat seedlings treated with 14α‐demethylase‐inhibiting fungicide, triadimefon. The results indicated that the BgABC1 was involved in the protection of the fungus against fungicide toxicity.     

Diagnostics of phytopathogen fungi <Emphasis Type="Italic">Septoria tritici</Emphasis> and <Emphasis Type="Italic">Stagonospora nodorum</Emphasis> by fluorescent amplification-based specific hybridization (FLASH) PCR

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola) and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.     

Evaluation of some Iranian wheat elite lines’ reaction to Septoria tritici leaf blotch

Septoria tritici blotch is an important wheat disease in many areas of the world including Iran’s warm, humid regions. In this study, reaction of 79 lines from elite regional wheat yield trials of the Cereal Research Department from four climate zones was evaluated to S. tritici leaf blotch in adult plant stage under an artificial field inoculation in Khuzestan province in the south of Iran for two years (2008–2009 and 2009–2010). Nurseries were artificially inoculated by spreading infected plant debris (from last growing season). Taking notes was performed with a modified Saari and Prescott method in 00–99 double-digit scale. Wheat lines were classified in groups of immune (00), highly resistant (11–14), resistant (15–34), moderately resistant (35–44), moderately susceptible (45–64), susceptible (65–84) and highly susceptible (85–99). Result showed that from 79 lines, 37 lines had susceptible reaction; 2 lines had moderately susceptible; and 40 lines showed immune.