Metabolic alteration induced by Botrytis allii in two onion cuitivars with different resistances to neck rot

Disease index, dry weight loss and leakage of K⁺ ions in the tissues of two onion cultivars demonstrated that the red cultivar Tropeana had a greater susceptibility to neck rot caused by Botrytis allii than the white cultivar Rovato. The comparison between the cultivars showed that the less susceptible one was characterized by lower polygalacturonase activity (with fewer isoenzyine forms), more rapid accumulation of phenolic compounds in the early stages and faster activation of peroxidase. Polyphenoloxidase was not found in either the healthy or the diseased tissues of the two cultivars. The significance of these findings is discussed in relation to possible host defence mechanisms.     

The effects of direct harvesting and drying systems on the incidence and control of neck rot (Botrytis allii) in onions

Direct harvesting, with mechanical removal of the foliage (topping), of onion crops followed by post-harvest drying at ambient temperatures ( c . 18°C) resulted in an increase in the incidence of onion neck rot ( Botrytis allii ).
The disease was substantially reduced if topped onions were dried at 30°C with an airflow of 425 m³ air/h/tonne. The treatment was most effective if the crop was removed from the field for drying within 48 h of topping thus avoiding severe infection of the damaged green tissues of the necks of onions.     

Sources, survival and transmission of Cladosporium allii and C. allii-cepae, leaf blotch pathogens of leek and onion

Neither Cladosporium allii nor C . allii-cepae was detected in seed samples of 46 commercial cultivars of leek and nine of onion.
In glasshouse tests where flower inflorescences of onions and leeks were dusted with conidia of C. alliicepae and C. allii , respectively, few onion seeds and no leek seeds became contaminated. In field crops of leeks some spathes and inflorescences became diseased and gave 0 002% C. allii contaminated seeds. No infected onion seeds were obtained from naturally-infected plants in the field.
Conidia of both fungi survived for less than 6 weeks in sterile and non-sterile soil (during which time the temperature varied from -8 to +11 C); pseudothecial bodies survived longer.
Both pathogens survived for 3 months in debris of onion or leek on the surface of soil in pots under similar conditions. In debris buried at a depth of 7 cm in sterile or unsterile soil they survived for 2 months. Although C. allii survived only 1 month in debris on the soil surfaces following a leek crop, conidia of the fungus were trapped from the air above the area for at least 6 months and healthy leeks transplanted into this area developed the disease. No leaf blotch developed in leeks grown on land which had not previously borne that crop.     

Further studies in increased sensitivity to N-phenylanilines in benzimidazole-resistant strains of Botrytis cinerea and Venturia nashicola

The ring-substituted N-phenylanilines, N-(3-chlorophenyl)aniline (MC-1) and N-(3,5-dichlorophenyl)aniline (MC-2) were tested for their antifungal activity against Botrytis cinerea Pers. ex Fr. and Venturia nashicola Tanaka et Yamamoto. In both fungi, increased sensitivity to MC-1 and MC-2 was clearly observed in ‘highly carbendazim-resistant, diethofencarb-sensitive’ (HR, S) phenotypes. Sensitivity was low in ‘carbendazim-sensitive, diethofencarb-resistant’ (S, R) and ‘intermediately carbendazim-resistant, diethofencarb-resistant’ (IR, R) strains. On cucumber cotyledons, other strains of B. cinerea, possessing the phenotype ‘highly carbendazim-resistant, diethofencarb-resistant’ (HR, R) were not controlled by either MC-1 or MC-2. Response to MC-2 was also examined using random ascospore progenies from V. nashicola crosses. In these progenies, high-level carbendazim resistance and MC-2 sensitivity always segregated together. Sensitivity to MC-2 is controlled by a single gene which is either identical to or very closely linked to one conferring high-level resistance to carbendazim.     

Negative cross-resistance of benzimidazole-resistant strains of Botrytis cinerea,Fusarium nivale and Pseudocercosporella herpotrichoides to various pesticides

Among benzimidazole-resistant strains ofFusarium nivale andPseudocercosporella herpotrichoides negative cross-resistance to N-phenylcarbamates like barban or chlorpropham was more common than to diethofencarb. Such differences were also observed with N-methylcarbamate or organophosphorus insecticides and with triazine herbicides. Several compounds belonging to these various groups of pesticides were highly toxic against the most common benzimidazole-resistant isolates ofBotrytis cinerea. They were not active, however, against isolates recently found in French vineyards that were treated with a mixture of diethofencarb and carbendazim. Some diphenylether derivatives seemed to be effective against all the benzimidazole-resistant phenotypes of this fungus.     

Effects of postharvest onion curing parameters on bulb rot caused by Pantoea agglomerans,Pantoea ananatis and Pantoea allii in storage

Crop loss of onion bulbs during storage carries an exceptionally high economic impact because a large portion of the production expenses has been expended before storage. Because of this, it is important to define practices that can reduce onion bulb losses caused by storage rots. This study investigates the impact of various curing parameters on disease development resulting from infection by Pantoea agglomerans, P. ananatis and P. allii on onion bulb cultivars Vaquero and Redwing, during storage. Overall, both the incidence and mean rot severity were similar amongst the bulbs under comparable conditions regardless of the species of Pantoea inoculated, although a significant difference was detected between the two onion bulb cultivars. In addition, a significant reduction of storage rot was observed when curing temperatures were ≤35°C. At temperatures >35°C, a shorter curing duration (2 days vs 14 days) decreased the severity of bulb rot due to Pantoea. This increased understanding of the inter‐relationships between the parameters used for curing, and the incidence and severity of bulb rot caused by Pantoea helps provide guidance towards using the curing process as a means to reduce the level of damage resulting from post‐harvest storage rot.     

Fungal development and plant response in detached onion, onion bulb scales and leaves inoculated with Botrytis allii, B. cinerea, B. fabae and B. squamosa

Fungal development and plant responses were examined in detached leaves and mid-bulb scales of Allum cepa. Following inoculation with suspensions of 105 conidia/ml distilled water Botrytis squamosa consistently produced spreading lesions in leaves and bulb scales. B. allii produced spreading lesions at most sites in bulbs but was very inconsistent in its infection of leaves; lesions were often confined to inoculation sites. Limited lesions were usually produced by B. cinerea but R. fabae failed to produce symptoms at most sites. Extensive colonization by B. allii and B. tauamosa required rapid penetration and totally necrotrophic fungal growth. During development of a spreading lesion, plant cell walls became very swollen around intramural hyphae and wall swelling appeared to precede epidermal cell death. Resistance to colonization was due to poor germination, failure to produce distinct infection hyphae (associated with accumulation of deposits of granular reaction material [RM] in underlying live cells) or restriction of infection ryphae amongst small groups of dead cells (limited lesion formation). Only B. fabae germinated poorly, and germ-tubes produced often failed to attempt penetration but grew over the leaf or bulb scale surface. Reducing numbers of conidia increased the frequency of sites associated with RM accumulation; granular deposits being particularly common at sites inoculated with low numbers of B. allii conidia. Electron microscopy revealed that RM granules were osmiophilic aggregates formed between the plasma membrane and epidermal cell wall. In the absence of RM, growth of avirulent species was restricted within the swollen walls of dead epidermal cells. Results ae compared with those from studies on tulip and broad bean leaves.     

Effect of temperature of production of Botrytis allii conidia on their pathogenicity to harvested white onion bulbs

Botrytis allii colonies incubated at low temperatures have been reported to produce larger conidia that germinate faster and give rise to longer germ-tubes than those grown at room temperature. The present study compared the effect of conidia produced at 20°C and at 0 and –2°C on their pathogenicity to artificially inoculated white onion bulbs, and the effect of conidial concentration (5×10³ and 5×10⁴ conidia/mL) on disease incidence, lesion area, incubation and latent period during storage at 20, 5 and 0°C. At all storage temperatures and periods tested conidia produced at −2°C caused a higher disease incidence and larger areas of rot than those produced at higher temperatures. When the conidial production temperature was raised to 20°C, the duration of incubation on the bulbs inoculated with 5×10⁴ conidia/mL was more than doubled during storage at 0°C, tripled at 5°C, and took 50% longer at 20°C. The incubation period was not significantly affected by conidial concentration at 20°C, and only slightly at 5 and 0°C, but at low temperatures the latent period was longer because of the delay induced in sporulation. These data are consistent with the packers' opinion that cross-infection of spring onions by long-term refrigerated onions in grading lines caused earlier and heavier rotting.     

Occurrence of bacterial rot of onion bulbs caused by Burkholderia cepacia in Japan

In 2001, a bacterial rot of onion (Allium cepa L.) bulbs was observed in Japan. The causal agent was identified as Bukholderia cepacia (Palleroni & Holmes 1981 ex Burhkolder 1950) Yabuuchi, Kosako, Oyaizu, Yano, Hotta, Ezaki, and Arakawa 1993. The identified bacteria were divided into two groups (Y and W) based on colony colors, and several phenotypic and genetic characteristics. Based on recA polymerase chain reaction assays, the strains of the Y and W groups belong to genomovar I (B. cepacia sensu stricto) and genomovar III (B. cenocepacia), respectively.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB162427 and AB162428     

Investigation of the host ranges of the leaf blotch pathogens of onion (Cladosporium allii-cepae) and leek (C. allii)

Inoculation of a range of Allium species and two non-alliaceous species with isolates of Cladosporiumallii-cepae and C. allii , obtained from onion and leek, respectively, demonstrated that the two pathogens had distinct host ranges. Conidia of C. allii-cepae, applied either dry or in aqueous suspension, infected A. altaicum, A. fistulosum (Japanese bunching onion), A. cepa (bulb onion), A. cepa var. ascalonicum (shallot), A. galanthum, A. pskemense and A. vavilovii . Dry conidia of C. allii applied at a high concentration caused atypical necrosis on A. altaicum, A. fistulosum, A. cepa var. ascalonicum, A. galanthum, A. pskemense, A. vavilovii, A. sativum (garlic), A. ampeloprasum and A. porrum (leek). Only A. ampeloprasum and A. porrum became typically infected following inoculation with conidia applied dry at low concentration or in aqueous suspension. Isolates of C. allii from leek failed to infect A. vineale, the type host. The length of conidia of a single isolate of C. allii-cepae varied significantly on different Allium spp.     

The composition of wall alterations and appositions (reaction material) and their role in the resistance of onion bulb scale epidermis to colonization by Botrytis allii

Ultrastructural examination of deposits of reaction material (RM) showed them to be composed of paramural granules (osmiophilic to varying degrees) lying within amorphous electron–dense material. Epidermal cell walls per se became increasingly electron–dense around the short infection hyphae produced by Botrytis allii. Fluorescence microscopy, histochemical tests and microautoradiography showed the accumulation of phenolic compounds at infection sites. Use of phenylalanine and cinnamate for E.M. autoradiography demonstrated the accumulation of phenolics within paramural granules, amorphous aggregates and within the penetrated cell wall, Paramural deposits were soluble in organic solvents and probably represented reservoirs of low MW phenolics which were progressively incorporated into insoluble polymers within the cell wall. Initial synthesis or accumulation of phenolics appeared to take place in rough endoplasmic reticulum during the first 2 h after cuticle penetration. Suppression of RM deposition and wall alterations was achieved by treatment of bulb scales with cycloheximide. Tissues treated with the protein synthesis inhibitor became susceptible to colonization by B. allii. Accumulation of phenolics at infection sites rendered the onion cell walls resistant to attack by a mixture of cell wall–degrading enzymes, Methanolic extracts of epidermal strips containing RM deposits contained very weakly active inhibitors of B. allii germ–tube growth. The role of RM deposits and wall alterations in the resistance of onion epidermis to colonization by Botrytis is discussed.     

The occurrence of postharvest gray-mold rot of sweet persimmon caused by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in Korea

In this study, we identified the causative agent of postharvest gray-mold rot in sweet persimmon fruit that were collected from Gangneung, Gangwon Province, Korea in October 2016. Symptoms included extensive growth of mycelia on post harvested fruit. The fungus was isolated from infected fruit and cultured on potato dextrose agar (PDA). For identification of the fungus, we examined morphology characteristics and rDNA sequencing analysis of the fungus and confirmed its pathogenicity according to Koch’s postulates. The results of morphological examinations, pathogenicity tests, 5.8S rDNA sequences of the internal transcribed spacer regions (ITS1 and ITS4) and the five nuclear protein-coding genes G3PDH, HSP60, RPB2, MS547 and TUB revealed that the causal agent of postharvest gray-mold rot on sweet persimmon fruit in Korea was Botrytis cinerea.     

Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.     

Soft rot of root chicory in Hokkaido and its causal bacteria

In August 2010, bacterial soft rot was found on root chicory (Cichorium intybus var. sativum) in Hokkaido, Japan. Severely infected plants in fields were discolored, had wilted foliage, and black necrosis of petioles near the crown. Wilted leaves subsequently collapsed and died, forming a dry, brown or black rosette. The root and crown became partially or wholly soft-rotted. Slimy masses on infected areas of roots, turned dark brown or black. Gram-negative, rod-shaped, peritrichously flagellated, facultatively anaerobic bacteria were exclusively isolated from rotted roots, and typical symptoms were reproduced after inoculation with the strains. The bacteria were identified as Dickeya dianthicola, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. odoriferum based on further bacteriological characterization and the sequence analysis of the malate dehydrogenase gene and 16S rRNA gene. These bacteria should be included with the previously reported Dickeya (=Erwinia) chrysanthemi in Saitama Prefecture, Japan, as causal pathogens of bacterial wilt of chicory.     

Interaction of Four Antagonistic Fungi with Botrytis aclada in Dead Onion Leaves: A Comparative Microscopic and Ultrastructural Study

ABSTRACT The colonization of dead onion leaves by Botrytis aclada and the fungal antagonists Aureobasidium pullulans, Chaetomium globosum, Glio-cladium catenulatum, and Ulocladium atrum and the interactions between B. aclada and each of the four antagonists were studied at the microscopic and ultrastructural level. This approach was used in an attempt to understand the colonization pattern of these fungi and the nature of the biocontrol activity of the antagonists that have shown a potential to suppress spore production of Botrytis spp. on necrotic plant tissues. When applied alone, B. aclada and U. atrum were found throughout the leaf tissues in high densities after an incubation period of 6 days at 18 degrees C in a moist chamber. C. globosum and G. catenulatum colonized only the outer portions of the leaf, whereas A. pullulans appeared to be concentrated in the leaf stomata. When pathogen and antagonists were applied together, ultrastructural observations revealed that cells of B. aclada were plasmolyzed in the presence of G. catenulatum, suggesting a reaction to antifungal molecules. Antibiosis also seemed to be involved, albeit to a lesser extent, in the antagonistic interactions between B. aclada and A. pullulans or C. globosum. No evidence of direct parasitism was recorded. On the other hand, U. atrum appeared to completely exclude B. aclada from dead onion tissues when both fungi competed for the substrate. Ultrastructural observations of the in vitro interaction between the two fungi did not reveal parasitism or antibiosis by either fungus. Based on previous records of its biocontrol potential and observations of its colonizing properties, it appears that U. atrum can compete for and utilize necrotic tissues rapidly and extensively, thus, excluding competitors without any other antagonistic action.     

Differential binding of a N-phenylformamidoxime compound in cell-free extracts of benzimidazole-resistant and-sensitive isolates of Venturia nashicola,Botrytis cinerea and Gibberella fujikuroi

Venturia nashicola isolates with a high level of resistance to carbendazim showed either increased sensitivity or were resistant to theN-phenylformamidoxime compoundN-(3,5-dichloro-4-propynyloyphenyl)-N′-methoxyformamidine (DCPF). Isolates with an intermediate or low level of carbendazim resistance were resistant to DCPF. Increased sensitivity to DCPF was also associated with a high level of carbendazim resistance inBotrytis cinerea but not with a moderate resistance level. Increased sensitivity to DCPF was not observed in carbendazim resistant isolates ofGibberella fujikuroi.Binding of [¹⁴C]DCPF in cell-free mycelial extracts of the highly carbendazim-resistant and DCPF-sensitive isolate ofV. nashicola was higher than in those of DCPF-resistant isolates that were either highly-resistant, intermediately resistant, or weakly resistant to carbendazim or were sensitive to carbendazim. [¹⁴C]carbendazim binding in extracts of highly carbendazim-resistant isolates ofB. cinerea was lower than that in extracts of sensitive isolates, whereas [¹⁴C]DCFP binding was higher. A decreased [¹⁴C]carbendazim binding was also observed in extracts of carbendazim-resistant isolates ofG. fujikuroi, binding of [¹⁴C]DCPF, however, was similar in extracts of both carbendazim-resistant and sensitive isolates.     

Volatile Metabolite Profiling for the Discrimination of Onion Bulbs Infected by Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii

The volatile metabolites of the headspace gas of onion bulbs inoculated with three different pathogens, Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii, were profiled using gas chromatography/mass spectrometry. Differences in the number and amount of volatile metabolites were observed. Two hundred and fifty three volatile metabolites were detected in bulbs inoculated with three pathogens or sterile distilled water. On day three, 202 volatile metabolites were observed, compared to 166 on day six. Of the 253 compounds, however, only 59 occurred relatively consistently over replications, of which 25 compounds were specific to one or more pathogens, including 10 that were unique to a pathogen. Metabolites such as 1-Oxa-4,6-diazacyclooctane-5-thione and 4-mercapto-3-(methylthio)--(thio-lactone)-crotonic acid were exclusive to onions inoculated with F. oxysporum. Acetone, acetic acid-hydrazide, propylcarbamate, 1-bromo-1-propene, thiirane, 1-(methylthio)-E-1-propene and 1-ethenyl-4-ethyl-benzene were specific to B. allii. 3-bromo-furan was specific to E. carotovora ssp. carotovora. Sterile water-inoculated bulbs produced 3,3-dioxy-1,2-propanediol-tetranitrate. Highest amount of sulfurs was found in pathogen-inoculated, while highest amounts of terpenes, aromatics and aliphatics were found in sterile distilled water-inoculated bulbs. The possible use of these differences in the volatile metabolites for detecting and discriminating diseases of onion in storage is discussed.