First report of celery stunt anthracnose caused by <Emphasis Type="Italic">Colletotrichum simmondsii</Emphasis> in Japan

A new disease was found in Japan, on celery (Apium graveolens var. dulce) having severe chlorotic leaf spot, stunt, and dwarf with leaf curl. A spore suspension from the fungus isolated from affected plants induced identical symptoms 14 days after plants were sprayed. Identification and molecular characterization showed that the causal agent is Colletotrichum simmondsii. This report is the first of stunt anthracnose on celery caused by C. simmondsii. We propose the name “stunt anthracnose” for the new disease. Colletotrichum acutatum sensu lato, as reference pathogen of celery anthracnose, should be changed to C. fioriniae based on morphological and molecular characteristics.     

Extracts from <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> induce effective resistance to the pathogen in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and solanaceous plants

Plants defend themselves against microbial invasions by detecting conserved molecules, collectively called pathogen-associated molecular patterns (PAMPs). PAMPs-triggered basal resistance is the first inducible layer of plant defense. Here we found that Ralstonia solanacearum strain RS1002 can efficiently grow and cause disease in ecotype Columbia-0 of the model plant Arabidopsis thaliana in a manner dependent on the Hrp type III secretion system (T3SS). The extent of disease symptoms caused by R. solanacearum was reduced in plants pretreated with ΔhrpY mutant deficient in the functional Hrp T3SS. Pretreatment with a boiled extract (BE) from R. solanacearum had a similar inhibitory effect on disease development or bacterial multiplication in both Arabidopsis and several solanaceous plants. Simultaneous inoculations with BE and R. solanacearum did not induce BE-mediated resistance, nor did a BE treatment with proteinases. These results indicate that host plants recognize an unknown proteinaceous PAMP in the BE to induce disease resistance and that the Hrp T3SS of R. solanacearum can suppress it. From an analysis using Arabidopsis mutants lacking PAMP receptors, the elongation factor Tu of R. solanacearum was shown to partially contribute to BE-mediated basal resistance in Arabidopsis plants.     

Plectosporium blight of monkshood caused by <Emphasis Type="Italic">Plectosporium tabacinum</Emphasis>

Severe blight of potted seedlings of monkshood caused by Plectosporium tabacinum was found in glasshouses in Kagawa Prefecture in southwest Japan in May 2001. Root rot and browning of stem bases were followed by wilting and yellowing of leaves, then blighting of leaves, flower buds and stems. A fungus was isolated from diseased plants and confirmed to cause the disease. The new disease was named “Plectosporium blight of monkshood”.     

A broad spectrum,one-step RT-PCR to detect <Emphasis Type="Italic">Satsuma dwarf virus</Emphasis> variants using universal primers targeting both segmented RNAs 1 and 2

Universal primers to detect Satsuma dwarf virus (SDV), including distantly related strains Citrus mosaic virus (CiMV), Navel orange infectious mottling virus (NIMV), and Hyuganatsu virus (HV), were tested in a convenient one-step RT-PCR assay. SDV was the most broadly detected using uSDVup/uSDVdo primers that specifically targeted a nucleotide sequence in the 3′-noncoding region that is conserved in both segmented RNAs 1 and 2 of SDV among the tested primers. Nucleotide sequence analysis confirmed that the amplified RT-PCR products could be derived from RNAs 1 or 2 of SDV variants, some of which had interesting genetic diversity.     

<Emphasis Type="Italic">In silico</Emphasis> genomic subtraction guides development of highly accurate,DNA-based diagnostics for <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2 and blood disease bacterium

New rapid diagnostic methods are urgently needed to discriminate the quarantine pathogen Ralstonia solanacearum (Rs) race 3 biovar 2 (R3B2) from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. We used an in silico bioinformatic approach to identify several genome sequences potentially specific to R3B2 strains. Primer sets were designed to PCR-amplify sequences in these regions, and four sets were ultimately shown to be >99% accurate for detection of R3B2 strains. On the basis of these results, several primers were designed to enable development of a loop-mediated isothermal amplification assay that was rapid, technologically simple, and essentially 100% accurate for identification of R3B2 when applied to a comprehensive collection of geographically diverse Rs strains. We fortuitously found that a sequence in one of the “R3B2-specific” regions has ~90% identity to a sequence present in strains of the blood disease bacterium (BDB), a member of the Rs species complex that infects banana. Alignments of these sequences allowed design of a second PCR primer set that proved 100% accurate for identification of BDB strains when tested on the 22 BDB strains available to us. These results demonstrate the power of in silico genomic subtraction for rapid identification of population-specific DNA sequences and for the development of simple, reliable detection methods for Rs subpopulations.     

Using leaf tip necrosis as a phenotypic marker to predict the presence of durable rust resistance gene pair <Emphasis Type="Italic">Lr34</Emphasis>/<Emphasis Type="Italic">Yr18</Emphasis> in wheat

Fifty wheat varieties along with Jupateco-73 and Morocco were studied for the expression of leaf tip necrosis (LTN), a trait linked with the durable rust resistance gene pair Lr34/Yr18. LTN was frequent (i.e., ≥6) in nine replications of a field experiment over 3 years in 17 genotypes, and the varieties were considered positive for LTN. In molecular analyses of these varieties, having relative severity values up to 78 for yellow rust and 45 for leaf rust, the 150-bp Lr34/Yr18-linked allele was consistently amplified. Expression of LTN in six of nine replications is an appropriate threshold for predicting the presence of Lr34/Yr18 gene pair, and genotypes can be selected using this trait.     

Co-transmission of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> (TYLCV)-Mld and TYLCV-IL by the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The ability of the whitefly Bemisia tabaci to transmit two strains of Tomato yellow leaf curl virus, the Israel and Mild strains, was studied after serial transfers of individual whiteflies that were viruliferous for both strains to tomato plants. After single whiteflies had successive acquisition feedings first on a single plant infected with one strain and then on a plant infected with the other strain, the single whiteflies later transmitted intermittently one, the other, or both strains to the test plants during serial transfers at 1-day intervals. Because both strains were found in the head, abdomen, and legs dissected from whiteflies during the retention period after the two successive acquisition feedings, both strains apparently circulate from midgut cells to salivary glands through the hemolymph.     

The presence of double-stranded RNAs in <Emphasis Type="Italic">Alternaria alternata</Emphasis> Japanese pear pathotype is associated with morphological changes

Strains of the Japanese pear pathotype of Alternaria alternata were screened for double-stranded RNAs (dsRNAs). Four strains had several dsRNAs; strain N18 was associated with several dsRNAs and had impaired growth phenotypes such as irregular mycelium and abnormal pigmentation. We isolated dsRNA-cured isolates from strain N18 by single-conidium isolation. The dsRNA-cured isolates had recovered normal growth and pigmentation. Enlarged vesicles were observed in mycelial cells of the original dsRNA-carrying N18 strain. DAPI nuclear staining revealed regression of the nuclei in dsRNA-carrying N18 cells. These results indicate that the dsRNAs might have negative effects, such as apoptosis-like cell death, on the host fungus.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.