Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

Overexpression of <Emphasis Type="Italic">SlARG2</Emphasis> or <Emphasis Type="Italic">SlTD2</Emphasis> in Arabidopsis enhances resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> L.

Tomato (Solanum lycopersicum L.) ARGINASE2 (ARG2) and THREONINE DEAMINASE2 (TD2) are involved in plant defense. These enzymes act in the midgut of herbivores fed on tomato plants to degrade the essential amino acids Arg and Thr, respectively. Although it has been demonstrated that overexpression of the SlARG2 gene in tomato enhanced its resistance against M. sexta larvae, knock-down the expression of SlTD2 reduced the resistance of tomato to lepidopteran herbivores; it remains unclear whether overexpression of SlTD2 could enhance the resistance of the host plants to herbivores, or whether combined overexpression of SlARG2 and SlTD2 could lead to synergistically enhanced resistance to insects. Here, we generated transgenic Arabidopsis plants overexpressing SlARG2 (SlARG2 OE) and SlTD2 (SlTD2 OE) individually as well as in combination (SlARG2-SlTD2 OE). Overexpression of these genes did not affect Arabidopsis development, seed yield, or Arg and Thr content. Insect-feeding bioassay was performed by feeding diamondback moth (Plutella xylostella L.) larvae on detached leaves of wild-type, SlARG2 OE, SlTD2 OE, and SlARG2-SlTD2 OE plants. Larvae fed on SlARG2 OE leaves showed approximately 31% to 35% reduction in weight and 6% to 10% reduction in survival rate compared to those fed on wild-type leaves. Although larvae fed on SlTD2 OE leaves showed no reduction in survival rate, they gained less weight. Whereas larvae fed on SlARG2-SlTD2 OE leaves showed neither reduction in weight nor reduction in survival rate. We further investigated the arginase enzymatic activity of the SlARG2 OE and SlARG2-SlTD2 OE transgenic plants. The SlARG2 OE line most resistant to diamondback moth larvae displayed the highest arginase activity. Our data indicate that overexpression of SlARG2 or SlTD2 in Arabidopsis can enhance its resistance against diamondback moth, whereas combined overexpression of SlARG2 and SlTD2 did not generate synergistically increased resistance to diamondback moth.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> species in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) plantations in Mexico

The aim of this study was to identify the Colletotrichum species associated with anthracnose symptoms in coffee (Coffea arabica L.) plantations in northern Puebla, Mexico. In 2013, five surveys were conducted in different production areas and at different altitudes. Symptomatic leaves, shoots, and ripe and unripe fruits of the coffee variety Red Caturra were collected. Isolates were obtained and the Colletotrichum species were identified morphologically and characterized by multilocus sequence analyses of the ACT, CAL, GAPDH, and TUB2 genes and the rDNA region. Additionally, pathogenicity tests were conducted using six isolates. We identified C. gigasporum, C. gloeosporioides, C. karstii (two isolates), C. siamense, and C. theobromicola. This is the first report of these five species infecting leaves of coffee. The symptoms caused by these species were characterized, but the species causing Coffee Berry Disease was not found. This is the first report of a complex of species affecting coffee plants in the same geographical area in Mexico, and suggests that other complexes of species may be important pathogens in coffee-producing areas elsewhere.     

The chemotaxis regulator <Emphasis Type="Italic">pilG</Emphasis> of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> is required for virulence in <Emphasis Type="Italic">Vitis vinifera</Emphasis> grapevines

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce’s disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce’s Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.     

Chitin elicitor receptor kinase 1 (CERK1) is required for the non-host defense response of <Emphasis Type="Italic">Arabidopsis</Emphasis> to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">cubense</Emphasis>

Banana wilt disease is a typical vascular disease caused by the fungal pathogen Fusarium oxysporum f. sp. cubense 4 (Foc 4). Pattern recognition receptors in the plant cell membrane can recognize pathogen-associated molecular patterns (PAMPs) to activate multi-layer defense responses, including defense gene expression, stomatal closure, reactive oxygen species (ROS) burst and callose deposition, to limit pathogen growth. In the present study, we found that chitin elicitor receptor kinase 1 (CERK1) was required for the non-host resistance of Arabidopsis thaliana to Foc B2 (a strain of Foc 4). The cerk1 mutant had weaker defense responses after Foc B2 treatment, including lower expression of PAMP- and salicylic acid-responsive genes, no stomatal closure, lower ROS level and less callose deposition, than that of the wild-type plant. Consistent with this, the cerk1 mutant plants exhibited higher susceptibility to non-host pathogen Foc B2. These results suggest the crucial importance of CERK1 in Foc B2-triggered non-host resistance.     

Rice <Emphasis Type="Italic">terpene synthase 18</Emphasis> (<Emphasis Type="Italic">OsTPS18</Emphasis>) encodes a sesquiterpene synthase that produces an antibacterial (<Emphasis Type="Italic">E</Emphasis>)-nerolidol against a bacterial pathogen of rice

Plant volatile compounds, including terpenes, are known to be involved in the rice defense system. In the present analysis of a terpene synthase, OsTPS18, in rice, we found that OsTPS18 was localized in the cytoplasm and synthesized the sesquiterpenes (E)-nerolidol and (E)-β-farnesene. The amounts of (E)-nerolidol and (E)-β-farnesene increased after jasmonic acid (JA) treatment. (E)-Nerolidol had significant antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that (E)-nerolidol plays an important role in JA-induced resistance against Xoo and that it functions as an antibacterial compound in rice.     

Antifungal activity of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. culture filtrates against the rice pathogen <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

An ethyl acetate extract of a culture filtrate (ECF) from an unidentified fungal isolate O821 was evaluated for antifungal activity against the rice pathogen Magnaporthe oryzae. The O821-ECF significantly inhibited spore germination, appressorium formation, and mycelial growth of M. oryzae, and its antifungal activity was heat-stable. It also significantly suppressed the number and size of blast lesions. In an analysis of the ITS sequence of this isolate, it shared similarities with species of the fungus Biscogniauxia. These results suggest that isolate O821 of the genus Biscogniauxia produces a heat-stable antifungal compound(s) in its culture filtrate.     

Postharvest evaluation of natural coatings and antifungal agents to control <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in <Emphasis Type="Italic">Rosa</Emphasis> sp.

Botrytis cinerea is a fungal pathogen that limits rose production and commercialization worldwide. Therefore, we evaluated a novel postharvest treatment against Botrytis cinerea in roses (Rosa sp. cv Vendela) using coating bases and antifungal agents of natural origin. Aloe vera pulp, cassava starch and gelatin were used as coating bases. Oregano essential oil (Origanum vulgare), thyme essential oil (Thymus vulgaris) and chitosan were used as natural antifungal agents. The coating bases were evaluated in different concentrations to observe effects of toxicity and opening diameter in rose buds. Gelatin and cassava starch coatings inhibited rose opening and showed petal damage in all concentrations tested. However, Aloe vera pulp at 25% allowed normal buds’ opening and no damage was observed, indicating that Aloe vera could be an ideal coating base for rose postharvest treatments. During in vitro assays, natural antifungal agents efficiently inhibited Botrytis cinerea growth in the concentrations tested. Further, mixture treatments of Aloe vera pulp (25%) with oregano essential oil (1%), thyme essential oil (0.1%) and chitosan (0.1%) showed independently neither damage nor opening inhibition in rose buds. Selected combinations of Aloe vera pulp and natural antifungal agents were applied in roses infected with Botrytis cinerea to evaluate their control of this pathogen. Unfortunately, the selected combinations did not reduce pathogen growth during postharvest treatments since they were similar to untreated controls. Further research has to be performed to find ideal combinations with Aloe vera that could inhibit B. cinerea during postharvest treatments in roses.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> associated with yield-decline of pyrethrum in Australia

Fusarium wilt, one of the destructive diseases of cucumber can be effectively controlled by using biocontrol agents such as Trichoderma harzianum. However, the mechanisms controlling T. harzianum-induced enhanced resistance remain largely unknown in cucumber plants. Here we screened the potent T. harzianum isolate TH58 that could effectively control F. oxysporum (FO). Glasshouse efficacy trials also showed that TH58 decreased disease incidence by 69.7 %. FO induced ROS over accumulation, while TH58 inoculation suppressed ROS over accumulation and improved root cell viability under F. oxysporum infection. TH58 inoculation could reverse the FO-induced cell division block and regulate the proportional distribution of nuclear DNA content through inducing 2C fraction. Moreover, the expression levels of cell cycle-related genes such as CDKA, CDKB, CycA, CycB, CycD3;1 and CycD3;2 in TH58 - pre-inoculated seedlings were up-regulated compared with those infected with FO alone. Taken together, these results suggest that T. harzianum improved plant resistance against Fusarium wilt disease via alterations in nuclear DNA content and cell cycle-related genes expression that might maintain a lower ROS accumulation and higher root cell viability in cucumber seedlings.     

<Emphasis Type="Italic">In vivo</Emphasis> evaluation of essential oils and biocontrol agents combined with heat treatments on basil cv Genovese Gigante seeds against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">basilici</Emphasis>

Seed treatments with essential oils (from savory and thyme) and biocontrol agents (Pseudomonas spp. and Fusarium oxysporum) have been evaluated in vivo after dry hot air treatments against Fusarium oxysporum f. sp. basilici on basil seeds. The savory and thyme essential oils showed a significant pathogen control activity because of their innate antifungal activity and because of the seed application method, but the dry hot pre-treatment did not show any obvious effect on the performance of the essential oil treatments. The dry heat treatment improved the Pseudomonas seed dressing effect against F.oxysporum f. sp. basilici, and showed important reductions in plant infection and the disease index on the treated seed plants, without any negative effect on seed germination. However, the pathogen control provided by the heat treatments combined with the application of the biocontrol agents never reached the same performance as the chemical treatments considered as the reference. Thus, short dry heat treatments on basil seeds have been shown to be a valid but complementary seed disinfection method against Fusarium wilt.     

Accumulation of toxigenic <Emphasis Type="Italic">Fusarium</Emphasis> species and <Emphasis Type="Italic">Stenocarpella maydis</Emphasis> in maize grain grown under different cropping systems

Entomopathogenic nematodes in the genus Steinernema are associated with Xenorhabdus spp. bacteria. When steinernematid colonise an insect host the nematode-bacterium association overcomes the insect immune system and kills the host within 48 h. Xenorhabdus spp. produce secondary metabolites that are antifungal to protect nematode-infected cadavers from fungal colonization. The concentrated, or cell-free metabolites of X. szentirmaii exhibit high toxicity against various fungal plant pathogens and show potential as natural bio-fungicides. In the current study, we determined 1) thermo-stability, 2) dose-response, and 3) shelf-life of antifungal metabolites of X. szentirmaii against Monilinia fructicola (cause of brown rot of peach and other stone fruit) and Glomerella cingulata (cause of antharacnose). Thermo-stability was determined by autoclaving bacterial culture broths (121 °C and 15 psi for 15 min) and measuring fungal growth on in potato dextrose agar (PDA) containing 10% of the supernatants. Autoclaving had no impact on the antifungal activity of the secondary metabolites. Over a test period of 9 months, the activity of both extract types did not decline when stored at 4 or 20 °C. A dose-response study (10, 20, 40, 60, 80 and 100% supernatant-containing metabolite) using both phytopathogens demonstrated that a greater dose of supernatant increased antifungal activity. The antifungal-metabolite containing supernatant of X. szentirmaii has potential as a bio-fungicide. These results demonstrate the metabolite(s) are thermo-stable, they have a long shelf-life and require no stabilizing formulation, even at room temperature.     

Rutin promoted resistance of tomato against <Emphasis Type="Italic">Xanthomonas perforans</Emphasis>

Xanthomonas perforans is the causal agent of bacterial spot, one of the most devastating diseases of tomato that results in considerable yield losses worldwide. Rutin, as a polyphenolic substance, was used to induce resistance in tomato against X. perforans. Rutin at concentration of 2 mM had ability to reduce the disease severity of bacterial spot. On the other hand, 2 mM rutin had no antibacterial activity in vitro. Expression profiling of pathogenesis-related gene 5 (PR-5), Phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) was probed during the enhanced resistance by rutin. Pretreatment with rutin (rutin/ X. perforans) led to induction of PR-5, PAL and LOX compared to controls (water/ X. perforans). Our results suggest that rutin-induced resistance against X. perforans in tomato might be mediated through stimulation of some defense genes such as PR-5, PAL and LOX.     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

Chitinolytic <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> E44G enhances the biocontrol efficacy against <Emphasis Type="Italic">Fusarium</Emphasis> wilt disease of tomato

Streptomyces griseorubens E44G is a chitinolytic bacterium isolated from cultivated soil in Saudi Arabia (a hot, arid climatic region). In vitro, antifungal potential of S. griseorubens E44G was assessed against the phytopathogenic fungus, Fusarium oxysporum f. sp. lycopersici (the causative agent of the Fusarium wilt disease of tomato). An inhibition zone of 24 mm was recorded. The chitinolytic activity of S. griseorubens E44G was proved when the colloidal chitin agar plate method was used. A thermostable chitinase enzyme of 45 kDa molecular weight was purified using gel filtration chromatography. The optimum activity was obtained at 60 °C and pH 5.5. The purified enzyme has shown a very pronounced activity against the phytopathogenic fungus, F. oxysporum. The molecular characterization of the chitinase gene indicated that it consists of 1218 bp encoding 407 amino acids. The phylogentic analysis based on the nucleotide DNA sequence and the deduced amino acids sequence showed high similarity percentages with other chitinases isolated from different Streptomyces species. In the field evaluation, application of both S. griseorubens E44G treatments significantly increased all tested growth and yield parameters and decreased the disease severity compared with the infected-untreated tomato plants suggesting potential as a biocontrol agent.     

Population features of <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">pruni</Emphasis> from <Emphasis Type="Italic">Prunus spp.</Emphasis> orchards in northern Italy

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italy. A set of 23 strains has been isolated in peach and plum orchards in an intensively stone fruit cultivated area located in north-eastern Italy. They were all identified as X. arboricola pv. pruni by means of phytopathological and serological features: hypersensitive reaction on bean pods, pathogenicity test on immature peach or plum fruitlets, identification by immunofluorescence assay and conventional PCR. Phylogenetic analysis based on sequencing of the gyrB housekeeping gene of the isolates showed that they formed a unique clade, well characterised and separated from other xanthomonads. An insight into the genetic population features was attempted by rep-PCR analysis, using the ERIC, REP and BOX primers. The combined rep-PCR fingerprints showed a slight intra-pathovar variation within our isolates, which grouped in five close clusters. Copper resistance has been assessed in vitro for our whole X. arboricola pv. pruni collection, highlighting that two isolates show a level of resistance in vitro up to 200 ppm of copper. Nonetheless, the copLAB gene cluster, present in many other species of Xanthomonads, was not detected in any isolate, confirming the presence of a still unknown mechanism of copper detoxification in our Xanthomonads arboricola pv. pruni tolerant/resistant strains.     

Host-delivered RNAi-mediated root-knot nematode resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis> by targeting <Emphasis Type="Italic">splicing factor</Emphasis> and <Emphasis Type="Italic">integrase</Emphasis> genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.     

Competence of <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Frankliniella intonsa</Emphasis> strains as vectors for <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis>

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.