Preliminary study of the fumigant toxicity of allyl alcohol to three stored-product insects

The toxicity of allyl alcohol as a fumigant was assessed for adults ofTribolium castaneum (Herbst.),Rhyzopertha dominica (F.) andSitophilus oryzae (L.). Fumigations were carried out in 3.4–1 glass containers at concentrations of 1–15 mg/l for 3 h. The most sensitive species wasS. oryzae and the most tolerant wasT. castaneum. A dose of 15 mg/l for 3 h was lethal to all three species tested. The feasibility of proposing allyl alcohol as a candidate fumigant is discussed. Publication of the Agricultural Research Organization.     

Effect of rapid and gradual increase of osmotic stress on survival of entomopathogenic nematodes

The effect of rapid and gradual exposure of entomopathogenic nematodes to osmotic stresses on the induction of a dormant state was determined with the nematodeSteinernema feltiae IS-6 infective juveniles (IJs). Rapid exposure of nematodes to glycerol at concentrations of 24% and 28% (w/w) caused the nematodes to enter a dormant state which was characterized by shrinking and impeded motility of all nematodes within 8 h. However, pre-exposure to gradually increasing glycerol concentrations of 5%, 10% and 18% at 4-h intervals resulted in dormancy after 4 h exposure to 24% glycerol. The total time of exposure to glycerol solution was 16 h in gradual osmotic stress. For nematodes exposed to 24% glycerol solution either rapidly or gradually, recovery occurred after 40 min in distilled water. Infectivity of osmotically stressedS. feltiae IJs was evaluated by two criteria, insect mortality and invasion rate. The assays indicated that infectivity of nematodes desiccated by rapid and gradual osmotic stresses was similar to that of fresh nematodes. Rapid exposure ofS. carpocapsae ‘All’,S. riobravis ‘Texas’,S. glaseri ‘NI’ andHeterorhabditis bacteriophora HP88 IJs to the 24% glycerol solution resulted in dormancy within 8 h. These treatments caused mortality of 48.4% and 11.7% amongS. glaseri Nl andH. bacteriophora HP88 IJs, respectively. Similar effects were observed when these nematode species were exposed to increasing osmotic stress of 5%, 10% and 18% at 6-h intervals. Under these same conditions, mortality ofH. bacteriophora HP88 andS. glaseri Nl IJs was 27.5% and 61.8%, respectively. http://www.phytoparasitica.org posting Sept. 29, 2004.     

Laboratory selection of resistance by the red flour beetle,tribolium castaneum (Herbst), to an atmosphere of low oxygen concentration

Adult populations of the red flour beetle,Tribolium castaneum (Herbst), were exposed for 40 generations to an atmosphere containing 99.5% N₂ and 0.5% O₂ at 95% RH, in order to select a strain resistant to the low oxygen concentration (LOC) atmosphere. Selection pressure was maintained at between 50% and 70% mortality. At the 40th generation comparison of sensitivity between the selected strain and the original non-selected strain indicated a resistance factor at the 50% mortality level (LT50) of x 5.2. However, throughout the selections, log-time against probit-mortality curves remained roughly parallel from generation to generation and the slope remained low. These findings indicate a multiplicity of genetic factors that at a high level of selection contribute together towards an adaptation of the insects to survival in the LOC atmosphere. Removal of selection pressure from a sub-population of the selected strain from the 13th to 21st generation revealed that resistance was partially retained with a decrease in resistance factor of 23%. Although the study revealed that the insects were able to develop a strain resistant to hypoxia, exposures were at 95% RH to minimize the desiccation effect. This does not reflect field situations, where ambient relative humidities are generally below 70%.     

Translocation of apple proliferation phytoplasma via natural root grafts – a case study

Apple proliferation (AP), a phytoplasma-induced disease of apple trees, was proven to be transmitted through infected grafting material and sap-sucking insects. To date there are little firm data on disease propagation in the field via natural root grafts. This question was thus addressed in the present case study by investigating trees of a 24-year old commercial apple orchard (‘Red Chief’ on MM 111), where the existence of root connections was discovered accidentally. After having displayed specific AP symptoms, nine trees were cut down and the stubs were infiltrated or brushed with glyphosate. Herbicide injury, however, remained not only restricted to the treated stubs, but also spread to approximately 50 neighbouring trees. Surprisingly, none of the pollinators (‘Granny Smith’ on M 9) growing interjacently and alternating between herbicide-damaged main crop trees was affected. Respective to the position of the nine AP-infected and glyphosate-treated cut stumps, four sections in the orchard were defined, from which a total of 122 trees was sampled and analysed using qualitative real-time PCR for detection of AP phytoplasma. The pathogen was found in 71.4% of ‘Red Chief’ trees with severe herbicide damage and 18.8% of trees with partial herbicide damage. None of the 31 investigated pollinators was AP-infected. Our data indicate that root connections seem to play a role for the spread of AP phytoplasma at least in older orchards and between trees on vigorous rootstocks.     

Occurrence of rocket larkspur witches’ broom caused by “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma asteris” in Japan

In October 2001, a disease of rocket larkspur (Cosolida ambigua (L.) P. W. Ball et Heyw), characterized by witches’ broom, yellows and virescence of flowers, was found in Yakage Town in Okayama Prefecture. Electron microscopy revealed the presence of phytoplasma-like bodies in the phloem of diseased plants. The causal phytoplasma was identified as “Candidatus Phytoplasma asteris” based on 16S rDNA sequence analysis, and demonstrated to be acquired by the leafhopper Macrosteles striifrons. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB258330.     

The antifeedant effect of neem seed kernel extract and azadirachtin on nymphs ofEyprepocnemis plorans

Acridids belonging to different species and families exhibit large differences in their response to neem components. In this context the antifeedant effect of a methanolic neem seed kernel extract (NSKE) and of azadirachtin (AZA) on fourth-instar nymphs of the acrididEyprepocnemis plorans Charpentier (Saltatoria:Acrididae) was investigated. Nymphs were offered either saccharose-impregnated filter paper disks or leaves of broad beans, treated with neem components. The amount of substrate consumed was determined by weighing the filter paper or by measuring the leaf area. On filter paper both NSKE and AZA were highly active down to the 10⁻⁴% treatment. In the leaf treatment, however, AZA was definitely more active than NSKE, with 100% deterrence at 10⁻⁴% and 10⁻²%, respectively. The methanolic NSKE was somewhat more active than the commercial preparation ‘Neemark’.     

Detoxification of α-tomatine by tomato pathogens Alternaria alternata tomato pathotype and Corynespora cassiicola and its role in infection

In tomato plants, α-tomatine, a steroidal glycoalkaloid saponin, inhibits fungal growth. Tomato pathogens that produce host-specific toxins, Alternaria alternata tomato pathotype causing Alternaria stem canker and Corynespora cassiicola causing Corynespora target spot, were investigated for sensitivity to α-tomatine. Although spore germination of A. alternata pathogenic and nonpathogenic to tomato and of C. cassiicola pathogenic to tomato was not affected by 0.1 mM α-tomatine, spore germination of C. cassiicola nonpathogenic to tomato was significantly inhibited. This result showed that A. alternata, regardless of its pathogenicity, and only the C. cassiicola pathogenic to tomato are resistant to α-tomatine. Germinating spores of A. alternata and C. cassiicola resistant to α-tomatine detoxified α-tomatine by degrading it to a less polar product. After inoculation of tomato leaves, spores of A. alternata and C. cassiicola nonpathogenic to tomato germinated and formed appressoria, but did not form infection hyphae in host tissues. When a host-specific toxin (CCT-toxin) produced by C. cassiicola pathogenic to tomato was added to nonpathogenic spores, colonization within leaves was observed in A. alternata, but not in C. cassiicola. On the other hand, when spores of C. cassiicola nonpathogenic to tomato were suspended in spore germination fluid of nonpathogenic A. alternata with α-tomatine detoxification activity, the fungus could be induced to colonize leaves in the presence of CCT-toxin. These results indicate that A. alternata tomato pathotype and C. cassiicola pathogenic to tomato detoxify α-tomatine during infection and that this detoxification is essential for host colonization by pathogens that produce host-specific toxins.     

Cloning of a specific DNA region of white top pathogen of pea and its detection by polymerase chain reaction

White top strain (WT strain) of Pseudomonas syringae pv. pisi (Ppi) is a variant strain causing white top disease of peas. The WT strain is distinguishable from common Ppi strains only by symptom expression chlorosis and whitening of apical shoots. To develop a specific detection method for the WT strain, we cloned a specific DNA region of the WT strain using transposon tagging. Five mutants defective in white top symptom expression were obtained. A part of the Tn5-flanking region was cloned and labeled as a hybridization probe. One clone, pAY3, gave two signal bands, one of which was detected from the genomic DNA of all the WT and the common Ppi strains; another was specific to WT strains. A restriction map of pAY3 showed that it contains two BamHI fragments; one is 5.0kb in length involving a part of Tn5, and the other is 1.5kb, did not carry Tn5, and may have been accidentally ligated into pAY3. The 1.5-kb band was subcloned as pAY13 and was used as a probe. It hybridized specifically to WT strains. These results suggested that the WT strains have a specific DNA region and that part of the region was successfully cloned. Sequence analysis of pAY13 showed that it is similar to part of nonribosomal peptide synthetases (NRPSs) genes. The deduced amino acid sequence of pAY13 suggested the existence of eight conserved motifs of NRPSs. WT strain-specific PCR primers, PS1 and PS2, were designed from the DNA sequence. These primers gave a specific amplification product of 981bp from both the genomic DNA and a direct cell preparation of WT strains. No specific amplicon was produced from Ppi strains that caused only water-soaked lesions or from strains of other P. syringae pathovars. A specific amplicon was not produced from four strains of the pea pathogen: P. marginalis pv. marginalis, P. viridiflava, Erwinia carotovora ssp. carotovora, Xanthomonas campestris pv. pisi. Using the primers, WT strain was detected from water-soaked lesions and green and white tissues without water soaking.The sequence reported in this paper has been deposited in the DDBJ database under accession no. AB117755     

京水菜种子SU1抗菌蛋白的纯化、稳定性和抑菌机制

为拓展抗菌蛋白在新型生物农药上的潜在应用价值,通过SP-Sepharose阳离子交换层析、Mone S阳离子交换层析和Superdex 75凝胶过滤层析从京水菜Brassica juncea var. multisecta种子中纯化得到一种分子量约为30 kD的SU1抗菌蛋白,采用纸片扩散法测定该抗菌蛋白的稳定性和抑菌活性,并采用荧光染色法分析其抗菌机制。结果表明,在pH为1～3和11～13条件下,SU1抗菌蛋白抑菌活性不受影响;40～80℃热处理和100 mmol/L Mn~(2+)、Al~(3+)、Zn~(2+)、Cu~(2+)、Fe3+、Pb~(2+)、Mg~(2+)、K~+和Cr~(3+)离子处理后,该抗菌蛋白仍具有抑菌活性,而100 mmol/L Ca~(2+)离子处理后该抑菌蛋白的抑菌活性消失。此外,乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)溶液处理后SU1抗菌蛋白的抑菌活性消失,再次加入Cr~(3+)离子时,其抑菌活性恢复。SU1抗菌蛋白能抑制7种植物病原真菌的生长,可破坏尖孢镰刀菌Fusarium oxysporum菌丝细胞膜透性和细胞核的完整性,并引起线粒体膜电势增加和菌丝细胞脱氧核糖核酸的分解。     

Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subsp. carotovora

To elucidate the role of flagella in biofilm formation by Erwinia carotovora subsp. carotovora EC1, we used a nonflagellate, nonmotile mutant (ΔfliC) and a flagellate, nonmotile mutant (ΔmotA). A biofilm-inducing medium, which contains the yeast peptone (YP) medium plus the salts of M-63 minimal medium, supported biofilm formation to a greater extent than either the YP or Luria Bertani (LB) medium alone. We demonstrated that both the ΔfliC and ΔmotA mutants greatly reduced their ability to form a biofilm on the surface of the wells of polyvinyl chloride (PVC) microtiter plates. The inability of both mutants to form biofilm on the PVC surface was further confirmed with phase-contrast microscopy. Both aflagellate (ΔfliC) and flagellate (ΔmotA) nonmotile mutants were equally defective in attachment to the PVC surface. The treatment of bacteria with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which inhibits the motility of this organism, reduced greatly the biofilm formation. Based on these results, flagella-mediated motility may play an important role in biofilm formation of E. carotovora subsp. carotovora EC1.     

Occurrence of chrysanthemum virescence caused by “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia” in Okinawa

In 1999, a disease of chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura], characterized by virescence of flowers, occurred in Okinawa Prefecture. The causal agent was identified as “Candidatus Phytoplasma aurantifolia” based on 16S rDNA sequencing. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB247462.     

Effect of sugars and non-nutritive sugar substitutes on consumption of apple leaves by codling moth neonates

Recently we reported that monosodium glutamate stimulates feeding in neonates of the codling mothCydia pomonella (L.). Herein we extend our general knowledge about feeding stimulators in neonates of this species, by presenting the effects of several sugars (sucrose, glucose, fructose, and maltose) and non-nutritive sugar substitutes (Sweet’n Low® and Equal®) on consumption of apple leaf (HoneycrispTM) tissue. Glucose, fructose, maltose and aspartame-based Equal had no effect on leaf consumption. Sucrose at a high concentration significantly reduced leaf consumption and delayed commencement of feeding. Sweet’n Low at high concentrations significantly increased leaf consumption and accelerated the commencement of feeding. Saccharin hemicalcium salt was identified as an active ingredient of Sweet’n Low. At 500 ppm and 1000 ppm, saccharin hemicalcium salt increased leaf consumption and accelerated commencement of feeding. The practical aspects of our findings are discussed.     

Occurrence of strawberry marginal chlorosis caused by “Candidatus Phlomobacter fragariae” in Japan

In February 2004, a disease of strawberry (Fragaria × ananassa Duch.), causing little-leaf, proliferation, malformation of fruits, and marginal chlorosis of leaves, occurred in Ehime Prefecture, Japan. The causal pathogen was identified as a phloem-restricted bacterium-like organism, “Candidatus Phlomobacter fragariae,” based on polymerase chain reaction (PCR) detection, electron microscopy, and sequence analysis of PCR products. This is the first report of strawberry marginal chlorosis in Asia. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB246669.     

Induction of systemic resistance toColletotrichum falcatum in sugarcane by a synthetic signal molecule, acibenzolar-S-Methyl(CGA-245704)

The effect of a novel synthetic signal molecule, acibenzolar-S-methyl (CGA-245704; benzo [1,2,3] thiadiazole-7-carbothioic acid S-methyl ester), in inducing resistance in sugarcane against red rot disease caused by the fungusColletotrichum falcatum Went was studied. Application of CGA-245704 as a soil drench or along with marcotting rooting mixture induced resistance in sugarcane to challenge inoculation withC. falcatum. When the pathogen was inoculated by the plug method, it caused discoloration in the untreated control stalk tissues; however, in the stalk tissues pretreated with acibenzolar-S-methyl, pathogen colonization was considerably reduced. When the pathogen was inoculated by nodal swabbing, its penetration was arrested in the sensitized stalk tissues. An induced systemic resistance effect was found to persist up to 30 days in the pretreated cut canes. Increased phenolic content and accumulation of pathogenesis-related (PR) proteins,viz., chitinase, β-1,3-glucanase and thaumatin-like protein (PR-5), were observed in sugarcane plants treated with acibenzolar-S-methyl.     

Correlation between field disease and latent infection by Glomerella cingulata of strawberry plants as revealed by a simple diagnostic method using ethanol immersion

Leaves of strawberry plants growing in fields were collected and assayed by ethanol immersion treatment (SDEI) to detect latent infection by Glomerella cingulata and Dendrophoma obscurans. SDEI revealed that 83% of the plants in growers fields were latently infected by G. cingulata and 58% by D. obscurans. In such fields, only 0.8%–2.5% of the plants later became wilted or died because of G. cingulata. When the latently infected plants from naturally infested fields were kept under optimal conditions for disease development for 5 weeks, 70.0%–83.3% of them wilted or died from G. cingulata. However, only 5.6%–6.7% of the plants diagnosed by SDEI as being without latent infection wilted or died. The results indicate the importance of selecting healthy plants, suggesting that SDEI is an effective method for detecting latent infection and reducing losses from G. cingulata.     

Purification of lily symptomless virus. Use and value of antisera against intact and pyrrolidine-degraded virus for testing lilies and tulips

Lily symptomless virus (LSV) was purified by clarification with chloroform, precipitation with polyethylene glycol and NaCl, and differential centrifugation. The influence of the source material and some buffers on virus yields were determined.Antisera were prepared against intact and pyrrolidine degraded LSV. It was concluded that intact and degraded LSV have very few antigenic determinants in common or none at all. The sensitivities of the micro-precipitin test and the single immunodiffusion drop test were about the same, but lower than that of electron microscopy.In the testing of lilies for LSV the most reliable results in leaves were obtained during the period from two weeks after flowering until close to the end of the growing season, and in leaves growing at a level about one-fourth of the distance from the top of the stem. In contrast to secondary infections, primary infections were detected more successfully in stored bulbs than in leaves taken from plants in the preceding growing season.In the testing of tulips, LSV was detected better in flowers than in leaves. Detection of primary infections was almost impossible. Except for those with a pink flower, experimentally infected tulips remained symptomless.Samenvatting Het symptoomloos lelievirus (LSV) were gezuiverd door klaring met chloroform, precipitatie met polyethyleenglycol en NaCl en differentieel centrifugeren. Het effect van het uitgangsmateriaal en enkele buffers op de virusopbrengst werd nagegaan.Antisera werden bereid tegen intact en met pyrrolidine afgebroken LSV. Geconcludeerd were dat intact en afgebroken LSV geen of slechts enkele antigene determinanten gemeenschappelijk hebben.De gevoeligheid van de microprecipitatietoets en de enkele immuno-diffusiedruppeltoets is ongeveer gelijk. Met het elektronenmicroscoop kunnen echter lagere virusconcentraties worden aangetoond.Het toetsen van lelies op LSV gaf de betrouwbaarste resultaten met bladeren die op ongeveer driekwart hoogte van de stengel groeien en in de periode tussen 2 weken na de bloei en vrijwel het einde van het groeiseizoen worden geplukt. Primaire infecties konden, in tegenstelling tot secundaire infecties, beter worden vastgesteld aan bolmateriaal tijdens de bewaring dan aan bladeren in het voorafgaande groeiseizoen.Bij het toetsen van tulpen werd het LSV met grotere zekerheid vastgesteld in bloemen dan in bladeren. Het vaststellen van primaire infecties was bijna niet mogelijk. Na infectie van tulpen met LSV vertoonden alleen die met een rose bloemkleur symptomen.