Real-Time PCR Assay for the Diagnosis and Quantification of Co-infections by Diaporthe batatas and Diaporthe destruens in Sweet Potato.

Foot rot disease caused by Diaporthe destruens (formerly Plenodomus destruens) has become a major concern for the production of sweet potato [Ipomoea batatas (L.) Lam.] in Japan. A related fungus Diaporthe batatas, which causes dry rot disease of sweet potato, is native and is widespread in fields in Japan. The similar characteristics of these two pathogens pose a challenge for conventional disease diagnosis. Currently, there are no effective molecular measures for identifying and distinguishing D. destruens and D. batatas. Here, we demonstrate a real-time PCR assay that distinguishes and quantifies D. batatas and D. destruens from co-infected sweet potato. The assay was performed with various simulated DNA combinations of D. batatas and D. destruens ranging from 1:1 to 1:100000. The assay was also used with the ratios of D. batatas: D. destruens: sweet potato DNA ranging from 1:1:1 to 1:1:100000. These assays produced a specific amplification product for each of the pathogens, and quantified the fungal biomass over the entire range tested without detecting false positives. The assay was validated by using infected sweet potato collected from various fields; it showed sufficient sensitivity and specificity to quantify and distinguish D. batatas and D. destruens from these field samples. Thus, our real-time PCR assay would be a useful tool for diagnosis of D. batatas and D. destruens and is expected to provide the foundation for the design of integrated disease management strategies for foot rot disease in sweet potato.

One of two major paralogs of AVR-Pita1 is functional in Japanese rice blast isolates.

We analyzed the avirulence gene AVR-Pita1 in Japanese rice blast isolates to determine how they gain virulence toward rice cultivars containing the Pita resistance gene. An avirulent isolate, OS99-G-7a (G7a), from a Japanese commercial field contained two paralogs of AVR-Pita1, designated as AVR-Pita1(JA) and AVR-Pita1(JB). Analysis of virulent, independent mutants derived from G7a, a single avirulent progenitor strain, indicated that AVR-Pita1(JA) was functional but AVR-Pita1(JB) was nonfunctional. The most frequent mutation was loss of AVR-Pita1(JA). Analyses of field isolates collected from diverse areas in Japan revealed that most of the AVR-Pita1 genes carried by Japanese isolates were identical to AVR-Pita1(JA) or AVR-Pita1(JB). The relationship between these major paralogs in Japanese isolates and the virulence of the strains carrying them indicate that AVR-Pita1(JA) is functional but AVR-Pita1(JB) is not, as is the case in G7a. Isolates that show virulence toward rice cultivars containing the Pita gene are presumed to have evolved virulence from avirulent origins via loss of AVR-Pita1(JA), except for one case in which virulence resulted from a base substitution. In this study, we discuss the properties and specificities of Japanese rice blasts that relate to virulence against Pita-containing rice. Furthermore, we present a method to amplify AVR-Pita1(JA) and AVR-Pita1(JB) separately and, specifically, to monitor functional AVR-Pita1 in Japan.

Effects of carbon sources and amines on induction of trichothecene production by Fusarium asiaticum in liquid culture.

Fusarium asiaticum infects cereal crops and produces trichothecenes such as deoxynivalenol and nivalenol. To determine the trichothecene induction mechanism, effects of carbon sources on the production of deoxynivalenol, nivalenol, 3-acetyl deoxynivalenol (3ADON), and 4-acetyl nivalenol (4ANIV) were examined in liquid cultures incubated with various strains. Sucrose supported significantly higher levels of acetylated trichothecene production in all strains than did the other carbon sources. Structural isomers of sucrose did not induce trichothecene production. The inducing effect of sucrose on trichothecene production was lost after the carbon source in the culture medium changed from sucrose to maltose in the process of incubation. Tri4 and Tri5 expressions were specifically up-regulated in the sucrose-containing medium and down-regulated with sucrose exhaustion. These findings suggest that F. asiaticum recognizes sucrose molecules and regulates Tri gene expression and trichothecene production. Moreover, an accelerating effect on trichothecene production by acidification of the culture medium containing specific amines during fungal incubation was exhibited only in the presence of sucrose in the medium. F. asiaticum induces trichothecene production in the presence of sucrose and accelerates the production when the medium containing specific amines is acidified during incubation.

Suppression of a phospholipase D gene, OsPLDbeta1, activates defense responses and increases disease resistance in rice.

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice.

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.