Rapid detection ofRhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.     

Pathogenicity and symptom expressions of Rhizoctonia solani,R. oryzae and R. oryzae-sativae to some commercial cultivars of rice in Myanmar

Rhizoctonia complex of rice has been detected in rice growing areas of Myanmar. The primary objective of this study is to study the varietal response of rice to Rhizoctonia complex and to distinguish the symptom expression of rice responses to these pathogens. Myanmar rice cultivars namely Manawthukha, Shwethweyin, Sinthwelatt and Yezinlonthwe were used to inoculate with three isolates of each species of Rhizoctonia solani, Rhizoctonia oryzae and Rhizoctonia oryzae-sativae. The symptoms created by each species of Rhizoctonia were distinguished by the size and colour of the lesion. Variation in lesion length was observed among different isolate-rice cultivar combination. Shwethweyin variety is the most susceptible one to all the tested three species among the four tested varieties.     

Phenotypic variation of Rhizoctonia oryzae in sheath disease of rice in Myanmar

Similarity in the cultural characteristics among closely related species of Rhizoctonia creates confusion and uncertainity in diagnosis. The present research was conducted to study the existence of phenotypic groups among isolates of R. oryzae in Myanmar. It was aimed to study the variation in phenotypic and molecular profiles among some isolates of R. oryzae and R. zeae. We found the occurrence of two distinct phenotypic groups of R. oryzae and a group of R. zeae. Ribosomal DNA-ITS sequencing was conducted and the resulting dendrogram agreed with those of the morphological grouping. A genetic distance of 0.064–0.072 wts was found between the R. oryzae and R. zeae groups. A pairwise distance of 0.014 and 0.022 was found between the RO1 and RO2 groups of R. oryzae. Our research revealed the existence of two distinct phenotypes in the isolates of R. oryzae collected from rice sheath in Myanmar and their differentiating features with R. zeae.     

Recent progress and understanding of the molecular mechanisms of the rice–Magnaporthe oryzae interaction

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most devastating disease of rice and severely affects crop stability and sustainability worldwide. This disease has advanced to become one of the premier model fungal pathosystems for host—pathogen interactions because of the depth of comprehensive studies in both species using modern genetic, genomic, proteomic and bioinformatic approaches. Many fungal genes involved in pathogenicity and rice genes involved in effector recognition and defence responses have been identified over the past decade. Specifically, the cloning of a total of nine avirulence (Avr) genes in M. oryzae, 13 rice resistance (R) genes and two rice blast quantitative trait loci (QTLs) has provided new insights into the molecular basis of fungal and plant interactions. In this article, we consider the new findings on the structure and function of the recently cloned R and Avr genes, and provide perspectives for future research directions towards a better understanding of the molecular underpinnings of the rice–M. oryzae interaction.     

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.     

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp

Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus more costly and time‐consuming, or using only one fluorescent dye, thus less accurate. Methods and Results: A red primary fluorescence (autofluorescence) of the hyphal cell walls and septa of Rhizoctonia spp. with green excitation is evidenced in Rhizoctonia spp. This property is exploited and combined for the first time with a conventional DAPI fluorescence to accurately determine the nuclear condition of Rhizoctonia. This bi‐fluorescence imaging strategy depicted the nuclear condition in Rhizoctonia spp. more accurately than the conventional DAPI fluorescence used alone and was validated against isolates previously genotyped by DNA sequencing. Conclusions: We demonstrated that the bi‐fluorescence imaging strategy was safe, accurate and simple to perform and interpret. Significance and Impact of the Study: The developed bi‐fluorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia strains. Its simplicity is a key advantage when there are numerous cultures to be examined.     

Rhizoctonia spp. Associated with Container-Grown Ericaceous Plants in Scotland

Fungi with Rhizoctonia-like mycelia were isolated from the foliage, stem-base and roots of ericaceous plants collected from nurseries in Scotland. Isolated fungi were identified as either binucleate Rhizoctonia spp. or Rhizoctonia solani on the basis of hyphal characteristics and nuclear number. The optimum temperature range for growth of binucleate Rhizoctonia spp. and R. solani was 20 and 25 C, resepctively. All isolates tested for pathogenicity caused foliar browning, and webs of mycelial growth were observed on dead and dying foliage. Binucleate Rhizoctonia spp. and R. solani are recorded for the first time on container-grown ericaceous plants in Scotland.     

Development of a PCR Assay for the Molecular Detection of Phytophthora boehmeriae in Infected Cotton

Cotton blight, caused by the oomycete Phytophthora boehmeriae, is a serious disease of cotton in China. In wet weather conditions, P. boehmeriae is usually the primary pathogen, followed by many saprophytic fungi and pathogens such as Pythium spp., Fusarium spp., Rhizoctonia and others. As P. boehmeriae grows much slower than other pathogens, it is difficult to isolate and identify. A rapid and accurate method for its specific identification is necessary for the detection of blight in infected cotton tissue. The internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) from three isolates of P. boehmeriae were amplified using the polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. PCR products were cloned and sequenced. The sequences were aligned with those published of 50 other Phytophthora species, and a region specific to P. boehmeriae was used to construct the specific PCR primers PB1 and PB2. Over 106 isolates of 14 Phytophthora species and at least 20 other fungal species were used to check the specificity of the primers. PCR amplification with primers PB1 and PB2 resulted in the amplification of a product of approximately 750 bp only from isolates of P. boehmeriae. Using primers PB1 and PB2, detection sensitivity was approximately 10 fg DNA/μl. In inoculated plant material, P. boehmeriae could be detected in tissue 1 day after inoculation, prior to the appearance of symptoms. The PB primer‐based PCR assay provides an accurate and sensitive method for detecting P. boehmeriae in cotton tissue.     

Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Abstract

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.     

Interactions between Sarocladium oryzae and stem attacking fungal pathogens of rice

In laboratory tests Sarocladium oryzae, the sheath rot pathogen of rice was found to inhibit the mycelial growth of other stem-attacking rice pathogens. Among those inhibited, Sclerotium oryzae and Gaeumannomyces graminis var. graminis were most sensitive while Pyricularia oryzae and Rhizoctonia solani were less sensitive. Tissue-based tests made with rice culm segments established that Sarocladium oryzae inhibits mycelial growth and delays sclerotium formation in R. solani. Cerulenin, the toxin produced by Sarocladium oryzae showed a toxicity pattern towards rice pathogens similar to that of Sarocladium oryzae. The stem rot pathogen, Sclerotium oryzae was most sensitive to cerulenin. In two greenhouse experiments, IR58 rice plants inoculated with Sarocladium oryzae alone or together with Sclerotium oryzae, G. graminis var. graminis or R. solani were found to have reduced plant height and increased tiller number. Sheath rot severity increased when Sarocladium oryzae was inoculated as a single pathogen or together with others. Sheath rot inoculation reduced stem rot in rice plants by 76 and 58%, respectively, in Experiment 1 and 2. By its known antagonistic interaction towards stem rot and crown sheath rot pathogens which are sensitive to it and by other unknown interactions, sheath rot emerges as the dominant disease.     

A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions.     

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre‐existing and induced defence in rice

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss‐of‐function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild‐type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1‐overexpressing (GIF1‐OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild‐type plants. More importantly, higher levels of callose were deposited in GIF1‐OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1‐OE plants when compared with the wild‐type plants. We also found that defence‐related genes were constitutively activated in the GIF1‐OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.     

Root rot disease incidence and severity on some legume species grown in Samsun and the fungi isolated from roots and soils

Abstract

Root rot disease is very common in the bean, soybean, faba bean and pea plants growing areas in Samsun province. Disease incidence and severity were detected the highest at 93.8% and 55.4% in the bean growing area, and the lowest at 64.0% and 24.3% in the faba bean growing area respectively. In this study, a total of 2714 fungal isolates were obtained from some legume plants and soil samples. The most common fungi isolated from root and soil samples were Fusarium spp., multinucleate Rhizoctonia (MNR), binucleate Rhizoctonia (BNR) and Pythium spp. respectively. Fusarium spp. were isolated at high rates from all the examined areas. MN Rhizoctonia and BN Rhizoctonia were isolated both from inner and coastal areas of the province, whereas Pythium spp. were isolated in costal areas, except for the Vezirköprü district which is situated in the inner area. When looking at the interactions among pathogens causing root rot, it was found the great majority of the samples (30.4%) isolated both Fusarium spp. and MNR-BNR group fungi, whereas Fusarium spp. and Pythium spp. were isolated together from 10.9% of the samples and MNR-BNR and Pythium spp. from only 1.5% of the samples.     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background  

Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood.     

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX‐mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re‐sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

PCR‐based Rapid Assay for Discriminative Detection of Latent Infections of Rice Bacterial Blight

A triplex PCR method has been developed for the race‐specific detection of Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight (BB) pathogen of rice. For this, three primer sets were designed: for specific internal regions of two genes (hpaA and XorII very‐short‐patch‐repair endonuclease) and for a genomic locus derived from an amplified fragment length polymorphism (AFLP) fragment specific for the K3 and K5 races. The sizes of the PCR products when using XOOF/XOOR, XRMF/XRMR and XAF3F/XAF3R primer pairs were 327, 427 bp and 1 kb, respectively, when the assay was applied to detect the pathogen in solution and lesion exudates, and as a template. Amplicons were obtained without the need for any prior processing (e.g. DNA preparation from infected leaf or bacterial cell isolation from the lesion). Furthermore, the pathogen could be quickly detected in the asymptomatic rice leaf 3 days after inoculation and at a distance of 6 cm from the lesion site. This PCR‐based simple and rapid assay will be a useful method for the detection and identification of Xoo as well as for disease forecasting in paddy fields.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.