Preliminary characterization of race-specific elicitors from<Emphasis Type="Italic">Peronospora parasitica</Emphasis> and their ability to elicit phenolic accumulation in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Intercellular washing fluid (IWF) obtained from the susceptibleArabidopsis accession Ws-eds1 inoculated withPeronospora parasitica isolate Emoy-2, contained an elicitor of necrosis with ecotype specificity towardsArabidopsis accessions with particular resistance genes. This elicitor caused necrosis on the highly resistant accessions La-er, Nd-1 and partly on Col-5, but not on the susceptible accessions Ws-eds1 and Oy-0. In resistant plants, injection of IWF caused hypersensitive reaction (HR)-like cell collapse which was associated with the accumulation of phenolics and lignin-like material in walls of cells undergoing cell death. The elicitor is sensitive to proteinase K and pronase enzymes, heating and autoclaving but insensitive to periodate oxidation, freezing and thawing, and is not dialyzable. Results suggest that the elicitor is a protein. Fractionation experiments using size-exclusion membranes revealed that elicitor activity has a molecular weight in excess of 100 kDa. http://www.phytoparasitica.org posting July 13, 2003.     

Resistance to <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> Dierckx in garlic accessions

Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.     

Greenhouse and field screening of wild<Emphasis Type="Italic">Lycopersicon</Emphasis> germplasm for resistance to the whitefly<Emphasis Type="Italic">Bemisia argentifolii</Emphasis>

Thirty-two accessions of wild tomato (Lycopersicon spp.) germplasm were evaluated for resistance to the whiteflyBemisia argentifolii Bellows ⇐p; Perring in a greenhouse choice bioassay. Density data were recorded for the adaxial and abaxial leaf surfaces for (i) all life stages ofB. argentifolii and (ii) types I, IV, V and VI trichomes. Individual plant selections (33 from 22 wild tomato accessions) with high resistance were subsequently tested in the field to verify the resistance found in the greenhouse screening. Resistance was defined by the density of all life stages of the whitefly observed on the eight leaflets sampled at nodes 5 and 7. Only type IV trichomes had a consistent (but low) and significant negative correlation between trichome density and whitefly density for various life stages. The highest whitefly resistance was observed inLycopersicon pennellii accessions LA 716, LA 1340 and LA 2560. The most resistantL. hirsutum f.typicum accessions were LA 1777 and LA 1353. http://www.phytoparasitica.org posting Dec. 9, 2002.     

Note: Evaluation of antibacterial activity of essential oil of<Emphasis Type="Italic">Rosa damascena</Emphasis> on<Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Significant antibacterial activity was observed in the essential oil (E.O.) ofRosa damascena Mill. and the Minimum Bactericidal Concentration (MBC) of the E.O. was determined as 1386.5 μg ml⁻¹ forErwinia amylovora, the causal agent of fire blight disease. Ooze formation on immature pears and lesion formation in artificially inoculated shoots were completely (100%) prevented by the essential oil ofR. damascena (1500 μg ml⁻¹), essential oil ofThymbra spicata var.spicata (500 μg ml⁻¹) and streptomycin (100 μg ml⁻¹). Copper oxychloride plus maneb significantly reduced ooze formation and lesion formation, but the control was less than that obtained with the essential oils or streptomycin. The essential oil ofR. damascena may be a useful natural bactericide for the control of the fire blight pathogen,E. amylovora. http://www.phytoparasitica.org posting July 14, 2004.     

Insecticidal activity of crude seed extracts of<Emphasis Type="Italic">Annona</Emphasis> spp.,<Emphasis Type="Italic">Lansium domesticum</Emphasis> and<Emphasis Type="Italic">Sandoricum koetjape</Emphasis> against lepidopteran larvae

Crude ethanolic seed extracts ofAnnona muricata, A. squamosa (Annonaceae),Lansium domesticum andSandoricum koetjape (Meliaceae) collected from different locations and years in Maluku, Indonesia, were screened for inhibition of larval growth against the polyphagous lepidopteranSpodoptera litura (Noctuidae). Extracts ofA. squamosa were significantly more active (20-fold) than those ofA. muricata. A. squamosa collected from Namlea yielded the extracts with the greatest inhibitory activity. There were significant differences among locations for bothA. squamosa andA. muricata but not forL. domesticum andS. koetjape. Extracts ofA. squamosa, collected from Namlea, inhibited larval growth in a dose-dependent manner, with a dietary EC₅₀ (effective concentration to inhibit growth by 50% relative to controls) of 191.7 ppm fresh weight. Extracts ofA. squamosa collected from individual trees in Namlea also varied in growth inhibitory effect againstS. litura andTrichoplusia ni larvae. This species is a candidate for development of a botanical insecticide for local use in Indonesia. http://www.phytoparasitica.org posting Dec. 1, 2003.     

Induced resistance to<Emphasis Type="Italic">Cladosporium cucumerinum</Emphasis> in cucumber by pectinases extracted from<Emphasis Type="Italic">Penicillium oxalicum</Emphasis>

Pectinases extract (PE) from the fermentation product ofPenicillium oxalicum BZH-2002 was tested for its ability to induce protection against scab caused byCladosporium cucumerinum on cucumber (Cucumis sativus L.) plants. Seedlings with one true leaf were sprayed with various concentrations of PE (10–200 units ml⁻¹) 3 days before inoculation withC. cucumerinum. Results showed that the induced local protection against the pathogen was dose-dependent when the concentrations of PE were between 20 and 120 units ml⁻¹; systemically induced resistance against the pathogen was not observed. Boiled PE had a slight effect on disease reduction. Commercial pectinases prepared fromAspergillus niger showed lower protection against scab compared with PE when they were used at the same concentration of enzyme activity. No inhibitory activity was observed on conidial germination or germ-tube growth ofC. cucumerinum. PE was further evaluated for its enhancement of defense-related enzymes. Peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased in cucumber seedlings after treatment with PE. PO and PPO remained at a higher level in PE-pretreated seedlings throughout the experiment period whether pathogen-inoculated or non-inoculated, whereas PAL activity began to decrease 2 days after PE treatment. http://www.phytoparasitica.org posting July 14, 2004.     

Note: Tachinid parasitoids of<Emphasis Type="Italic">Ancyrosoma leucogrammes</Emphasis> and notes on parasitization rates of<Emphasis Type="Italic">Clytiomya dupuisi</Emphasis>

The study deals with the tachinid parasitoids ofAncyrosoma leucogrammes (Gmelin) (Heteroptera: Pentatomidae):Clytiomya dupuisi Kugler,Clytiomya sola (Rondani) andGymnosoma clavatum (Rohdendorf) (Diptera: Tachinidae). All species are recorded for the first time as parasitoids ofA. leucogrammes, andC. dupuisi was reared for the first time from a host. The parasitization rates ofC. dupuisi on adults ofA. leucogrammes varied from 7% to 9% between 1994 and 1999.C. sola andG. clavatum were reared in only small numbers. http://www.phytoparasitica.org posting July 10, 2003.     

Anthracnose of <Emphasis Type="Italic">Polygonatum falcatum</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum dematium</Emphasis>

Severe spotting, blight and drop of leaves caused by Colletotrichum dematium were found on potted plants of Polygonatum falcatum, a liliaceous ornamental, in open fields in Kagawa Prefecture, Japan, in May 2001. This new disease was named anthracnose of P. falcatum. Keisuke Tomioka, Jouji Moriwaki, Toyozo Sato contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under accessions MAFF239500 and AB334523, respectively.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Feeding behavior of<Emphasis Type="Italic">Aphis gossypii</Emphasis> on resistant accessions of different melon genotypes (<Emphasis Type="Italic">Cucumis melo</Emphasis>)

The feeding behavior of the melon aphidAphis gossypii Glover (Homoptera: Aphididae) was monitored using the electrical penetration graph (EPG) technique on different melon (Cucumis melo L.) genotypes showing resistance to the aphid. The aphid-resistant genotypes used were PI-161375 and PI-414723, sources of theVat andAgr genes, respectively. TGR-1551, a newC. melo accession from Zimbabwe, was also tested. Our goal was to localize the tissues where the resistance factors are expressed and to determine if the resistance mechanisms operating in the three aphid-resistant accessions were the same. Our results indicated that the three selected lines have resistant factors located at the epidermis, mesophyll and vascular tissues. However, the behavior ofA. gossypii on TGR-1551 was different from the two other resistant accessions, as indicated by a longer phloem salivation phase (E1 phase). Many of the E1 phases observed for aphids feeding on TGR-1551 were not followed by phloem ingestion (E2 phase). These results suggest that TGR-1551 has a resistance mechanism that preventsA. gossypii from initiating ingestion from the phloem. Preference tests under free choice conditions also showed that aphids rejected accessions TGR-1551 or PI-414723 faster than PI-161375. Our results support the hypothesis thatAgr andVat are coding for different kinds of resistance strategies. Comparisons of aphid life history parameters also indicated that TGR-1551 is a very promising new source to breed for resistance againstA. gossypii. http://www.phytoparasitica.org posting Jan. 16, 2002.     

A fungistatic chromene from<Emphasis Type="Italic">Ageratum conyzoides</Emphasis>

Ageratum conyzoides L. is an annual herb in the tropics and subtropics whose extracts are known to possess pharmacological and biocidal activity. We report on the bioactivity of a secondary metabolite (a chromene) isolated from the shoots ofA. conyzoides against some plant pathogenic fungi. Organic solvent extracts from the shoots were tested for antifungal activity against the plant pathogenic fungiRhizoctonia solani, Sclerotium rolfsii, Botryodiplodia theobromae, Phomopsis theae andFusarium species growingin vitro on potato dextrose agar medium. The cruden-hexane extract completely inhibited the growth ofR. solani andS. rolfsii. Then-hexane extract was chromatographed over a column of silica gel followed by activity-guided fractionation to give an antifungal principle. Structure elucidation by detailed analysis of¹H,¹³C NMR and mass spectroscopy identified the active compound as precocene II. The growth ofR. solani andS. rolfsii was completely inhibited by precocene II at a concentration of 80–100 ppm. The sclerotia ofR. solani andS. rolfsii were also completely suppressed by 150 ppm of precocene II. Sub-culture of these inhibited fungi onto precocene II-free medium restored growth of the fungus, indicating that precocene II is fungistatic. Crude or refined extracts fromA. conyzoides offer the possibility of biocontrol of plant pathogenic fungi. http://www.phytoparasitica.org posting Feb. 11, 2004.     

<Emphasis Type="Italic">In vitro</Emphasis> Selection of Maize Rhizobacteria to Study Potential Biological Control of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Flavi</Emphasis> and Aflatoxin Production

The aims of this study were to select bacterial isolates from the non-rhizophere of maize soil and to examine their antagonistic activity against Aspergillus section Flavi strains. The first selection was made through ecophysiological responses of bacterial isolates to water activity (a_w) and temperature stress. Subsequently, an Index of Dominance test (I_D), ecological similarity and inhibition of the lag phase prior to growth, growth rate and aflatoxin B₁ accumulation were used as criteria. From the first assay nine bacterial strains were selected. They grew well at 25 and 30 °C, with growth optima between 0.982 and 0.955 a_W using 48 h of incubation. There was ecological similarity between the bacterial strains Bacillus subtilis (RCB 3, RCB 6), Pseudomonas solanacearum RCB 5, Amphibacillus xylanus RCB 27 and aflatoxigenic Aspergillus section Flavi strains at 0.982 at 25 °C. The predominant interaction between all selected bacteria and fungi in dual culture was mutual intermingling at 0.982. Mutual inhibition on contact and mutual inhibition at a distance was observed at 0.955 a_w, between only four bacteria and some Aspergillus strains. Bacillus subtilis RCB 55 showed antifungal activity against Aspergillus section Flavi strains. Amphibacillus xylanus RCB 27, B.␣subtilis RCB 90 and Sporolactobacillus inulinus RCB 196 increased the lag phase prior to growth and decreased the growth rate of Aspergillus section Flavi strains. Bacillus subtilis strains (RCB 6, RCB 55, RCB 90) and P. solanacearum RCB 110 inhibited aflatoxin accumulation. Bacillus subtilis RCB 90 completely inhibited aflatoxin B₁ accumulation at 0.982 a_W. These results show that the bacterial strains selected have potential for controlling Aspergillus section Flavi over a wide range of relevant environmental conditions in the stored maize ecosystem.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Fumigant toxicity of<Emphasis Type="Italic">Eucalyptus blakelyi</Emphasis> and<Emphasis Type="Italic">Melaleuca fulgens</Emphasis> essential oils and 1,8-cineole against different development stages of the rice weevil<Emphasis Type="Italic">Sitophilus oryzae</Emphasis>

Essential oils extracted fromEucalyptus blakelyi (1,8-cineole, 77.5%),Melaleuca fulgens (1,8-cineole, 56.9%) and 1,8-cineole were shown to have fumigant toxicity against different development stages ofSitophilus oryzae. The eggs ofS. oryzae were the most tolerant, followed by pupae, larvae and adults in that order.M. fulgens oil,E. blakelyi oil and 1,8-cineole at 100 μl per liter of air gave, respectively, LT₅₀ values of 16.2, 17.4 and 9.1 h for adults, 31.1, 19.3 and 17.5 h for larvae, 55.6, 75.2 and 39.7 h for pupae, and required >7 days for eggs. Only 1,8-cineole (200 μl ⁻¹ air) gave a significant egg kill by 7 days and the LT₉₅ was 134.5 h. 1,8-Cineole could be a useful new fumigant. http://www.phytoparasitica.org posting Oct. 3, 2004.     

A quantitative PCR assay for accurate <Emphasis Type="Italic">in planta</Emphasis> quantification of the necrotrophic pathogen <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis>

A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.     

Comparative studies on the effects of a yucca extract and acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl (ASM) on inhibition of <Emphasis Type="Italic">Venturia inaequalis</Emphasis> in apple leaves

The effect of an extract of Yucca schidigera on the control and infection process of the apple scab pathogen, Venturia inaequalis, was examined and compared with the chemical resistance inducer, acibenzolar-S-methyl (ASM). In seedling assays, both materials significantly reduced apple scab symptoms and pathogen sporulation on leaves and both showed similar control efficacies as the reference treatment, sulphur. Whereas yucca extract and sulphur gave significant inhibition of conidial germination in vitro, ASM did not inhibit germination. Histopathological studies of the infection process of V. inaequalis in apple leaves showed that the yucca extract primarily acted by inhibiting pre-penetration events and penetration itself. In contrast, the ASM treatment significantly inhibited more stages of the infection process (pre-penetration, penetration and post-penetration events). These observations suggest that the yucca extract acted mainly by a direct fungitoxic effect whereas ASM, as expected, acted as a resistance inducer. However, expression studies of two genes encoding the PR proteins, PR1 and PR8, in apple seedlings indicated that yucca extract may also affect plant defence as expression of both genes was up-regulated following yucca treatment, to a level similar to that observed after treatment with ASM. The fungitoxic effect of sulphur on V. inaequalis was also confirmed in this study.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Daily consumption and predation rate of different<Emphasis Type="Italic">Stethorus punctillum</Emphasis> instars feeding on<Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Laboratory experiments were conducted to evaluate the prey stage preference and the daily consumption of each stage of the coccinellid predatorStethorus punctillum Weise (Coleoptera: Coccinellidae) feeding on the two-spotted spider miteTetranychus urticae (Koch) (Acari: Tetranychidae). Groups of different life stages of the prey were offered (eggs, larvae, nymphs and adults). The prey preference varied with the stage ofS. punctillum. First larval instars had no significant preference among theT. urticae stages offered. Second larval instars consumed significantly more spider mite larvae in comparison with nymphs. In contrast, third larval instars indicated a strong preference for mite eggs. Significantly fewerT. urticae larvae were consumed by the fourth larval instars ofS. punctillum, in comparison with the three other mite stages. Finally, adult predators consumed significantly more mite eggs than the other stages offered. This preferential trend was similar for all adults tested, whether during the pre-oviposition or the oviposition period. http://www.phytoparasitica.org posting Feb. 17, 2004.     

Tachinidae parasitoids of<Emphasis Type="Italic">Traumatocampa ispartaensis</Emphasis> from Turkey

Tachinid parasitoids ofTraumatocampa ispartaensis Doğanlar & Avcı (Lepidoptera, Thaumetopoeidae), which was found to be a new species, were collected from the cedar forests around Isparta-Kapıdağ. The species found wereBlondelia nigripes (Fall.),Compsilura concinnata (Meig.),Pales processioneae (Ratz.),Phryxe caudata (Rond.),Exorista segregata (Rond.) andCarcelia iliaca (Ratz.). Within the six species of Tachinidae,B. nigripes was the most common one, parasitizing up to 4.6% ofT. ispartaensis pupae. http://www.phytoparasitica.org