Novel methodologies in screening and selecting olive varieties and root-stocks for resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

An innovative inoculation process, involving the drilling of a trunk hole in 3 year-old olive trees and injecting a dense conidial suspension of Verticillium dahliae, was developed to study differentiation in foliar symptom expression between olive cultivars tolerant or susceptible to the pathogen. It was demonstrated that V. dahliae conidia could be translocated and colonize the xylem at the same distance above and below the point of trunk injection in both cultivars. However, the pathogen could be subsequently isolated at statistically significant percentages in susceptible cv. Amphissis compared to the tolerant cv. Kalamon, indicating operation of resistance mechanisms in the vascular phase of the disease. Consequently symptom development in the susceptible cultivar was at least sixfold more intensive compared to the tolerant cultivar, 6–11 months after trunk inoculation. Perennial olive orchard experiments, aimed at selecting Verticillium-resistant root-stocks, were conducted by applying the novel method in 2–3 year-old root-stock suckers of Amphissis olive trees and in the tolerant cvs Lianolia of Corfu and Koroneiki. It was indicated that potentially resistant root-stocks could be obtained following the trunk drilling technique. Resistance differentiation between cvs Amphissis and Kalamon was further verified through root inoculation by various V. dahliae microsclerotial concentrations and demonstrated that the trunk drilling inoculation procedure is equally efficient in resistance evaluation of olives to Verticillium wilt. The trunk inoculation procedure could be useful in selecting and screening root-stocks for resistance to V. dahliae and other vascular pathogens and could elucidate resistance mechanisms in woody plants against vascular wilt diseases.     

Control of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> at a strawberry nursery by paddy-upland rotation

Effects of crop rotation between rice paddy fields and strawberry nurseries on the control of Verticillium wilt of strawberry were studied. For detecting Verticillium dahliae, the causal agent of Verticillium wilt, in soil, eggplant was used as an indicator plant. We were thus able to detect as low as 1 microsclerotium/g dry soil. In field surveys of Chiba and Hokkaido from 2000 to 2003, V. dahliae was detected in 9 of 10 upland fields but in none of 21 paddy-upland fields. In Hokkaido during 2000–2007, strawberry mother plants were planted, and plantlets were produced in upland and paddy-upland fields to assess V. dahliae infestation. Verticillium wilt of strawberry had never occurred in 72 tested paddy-upland fields, compared to 13.2–73.9% of plantlets infected with V. dahliae in upland fields. In a pot experiment in a greenhouse, two flooding treatments or two paddy rice cultivations suppressed Verticillium wilt symptoms on eggplant. In field experiments, one paddy rice cultivation in Chiba and two in Hokkaido prevented development of Verticillium wilt symptoms on eggplant. Verticillium wilt of strawberry was controlled completely with one paddy rice cultivation in infested fields in Chiba. In these field experiments, the number of microsclerotia of V. dahliae decreased under the flooding conditions for paddy rice cultivation. Based on the reduction in microsclerotia, a crop rotation system with paddy rice for 3 years (three times), green manure for 1 year, and strawberry nursery for 1 year was designed for Hokkaido.     

Seedborne fungi detected on stored solanaceous berry seeds and their biological activities

We isolated 629 fungi from 1296 berry seeds of solanaceous plants, including tomato (Lycopersicon esculentum), eggplant (Solanum melongena), bell pepper (Capsicum annuum), and red pepper (Capsicum annuum var. annuum) preserved for long and short terms. The isolates were classified into 22 genera excluding unidentified fungi, and the fungal floras were divided into two types: the tomato–eggplant and pepper groups. The results of cluster analysis with unweighted pair-group method with arithmetic average also supported these groups. Most tomato seeds infested with Geotrichum candidum germinated and grew the same as uninfested seeds. Cladosporium sphaerospermum and Arthrinium sp. isolated from eggplant seeds strongly suppressed germination, and Penicillium variabile suppressed seminal root elongation on eggplant. Alternaria alternata, Botrytis cinerea, and Myrothecium verrucaria detected from red pepper or bell pepper seeds were pathogenic to the fruits and the seedlings after artificial inoculation.     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.     

Effect of plant roots on the germination of microsclerotia ofVerticillium dahliae

Induction of germination of microsclerotia by exudates from plant roots may be important for the control ofV. dahliae. Laboratory experiments with root observation boxes were carried out to assess the influence of root tips of seven crop species and cultivars on the germination of microsclerotia ofVerticillium dahliae in soil under controlled conditions. The root density of crops was measured in a field experiment. The results of the laboratory experiments and the field experiment were combined to estimate the total effect of crops on the population of microsclerotia in the field. Germination of microsclerotia was stimulated by all crops compared to a control without a crop. Among crops, roots of potato cvs Element and Astarte had a larger stimulation effect on microsclerotia than that of potato Ostara, pea, flax, sugar beet or onion. The number of hyphae per microsclerotium decreased with distance from the root surface regardless of the crop species or cultivar. Differences in root densities, in the affected root zones and in the stimulation effect on germination of microsclerotia caused large differences among crops in the effect on the population of microsclerotia in the soil. However, growing a rop with the special purpose to reduce the level ofV. dahliae inoculum in the soil is an inefficient control measure, because only a small part of the total soil volume is affected by roots and the number of hyphae per microscleroium affected is too low.Abbreviations MS microsclerotia, microsclerotium     

Weed hosts of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in cotton fields in Turkey and characterization of <Emphasis Type="Italic">V. dahliae</Emphasis> isolates from weeds

A weed survey conducted in 2004 and 2005 in Aydin province of Turkey showed that Solanum nigrum, Xanthium strumarium, Amaranthus retroflexus, Portulaca oleracea, Sonchus oleraceus and Datura stramonium were the most prevalent weeds in the cotton fields exhibiting Verticillium wilt. Verticillium dahliae Kleb. was recovered from A. retroflexus and X. strumarium in those cotton fields. This is the first report of V. dahliae occurring naturally in A. retroflexus in Turkey. Pathogenicity tests on cotton and weeds showed that the virulence of V. dahliae isolates from weeds was higher on cotton plants than on weeds, with the disease severity ranging from 31.7% to 98.0%. Disease severity of V. dahliae isolates was 54.7–93.9% on eggplant, 23.7–51.6% on cucumber and 11.0–16.4% on tomato, whereas it did not cause any disease symptoms, or only low levels, on pepper and bell pepper. Two vegetative compatibility groups (VCGs) were identified among seven tested weed isolates: VCG2A (two isolates) and VCG2B (three isolates) using international reference strains.     

Extracts of killedPenicillium chrysogenum Induce resistance against Fusarium wilt of melon

Dry fungal biomass ofPenicillium chrysogenum (dry mycelium), a waste product of the pharmaceutical industry, was extracted with water and applied to the roots of melon plants before or after inoculation withFusarium oxysporum f.sp.melonis (Font). Seedlings (4–6 days after emergence) treated with either acidic dry mycelium extract (DME) or neutralized dry mycelium extract (NDME) were protected against challenge infection withFom. A single drench with 2–5% DME applied 12–72 h before inoculation provided significant control of the disease compared with water-drenched, challenged seedlings. No protection was seen in plants treated 0–6 h before inoculation or 0–48 h after inoculation. Neither DME nor NDME (0.5–5%) had any effect on fungal growthin vitro, which implied that disease controlin vivo was mediated by induced resistance. The resistance induced by DME protected melon plants not only against race 1,2, but also against the three other races of the pathogen, indicating a race-non-specific resistance againstFom. Both DME and NDME significantly increased peroxidase activity and free L-proline content in seedlings 12 h and 48 h after soil drench, respectively. Resistance to Fusarium wilt was significantly associated with elevated levels of peroxidase activity but not with free L-proline content. Thus, peroxidase might be involved in the defense mechanisms activated by DME or NDME. http://www.phytoparasitica.org posting Aug. 31, 2001.     

Suppression of Verticillium Wilt in Eggplant by Some Fungal Root Endophytes

One hundred and twenty-three fungal isolates were obtained from 225 root segments of eggplants, melon, tomato, strawberry and Chinese cabbage, grown as bait plants in a mixed soil made up of samples from different fields in Shizuoka, Japan. Isolates belonging to Mycelium radicis atrovirens (MRA), including Phialocephala fortinii, were the most prevalent in all the five bait plants. Eleven of the 123 isolates, after being inoculated onto axenically reared eggplant seedlings, almost completely suppressed the pathogenic effects of a post-inoculated, virulent strain of Verticillium dahliae. Seven of these 11 isolates had come from the roots of eggplant and included Heteroconium chaetospira, P. fortinii, and unidentified species of Fusarium, Penicillium, Trichoderma and MRA. P. fortinii, H. chaetospira, a non-sporulating isolate with white mycelium (SWM) and MRA were easily reisolated from root segments. Hyphae of H. chaetospira, P. fortinii and SWM colonized the root tissues of eggplant without causing apparent pathogenic symptoms. The mechanisms by which these endophytes confer resistance to infection by V. dahliae are unknown but the effectiveness of these fungi in a laboratory setting indicates that they have potential as biocontrol agents and merit further investigation.     

Incidence of Verticillium wilt of artichoke in eastern Spain and role of inoculum sources on crop infection

Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield. http://www.phytoparasitica.org posting July 21, 2005.     

Induced resistance of cucumber seedlings caused by some non-pathogenicRhizoctonia (np-R) isolates

Among 153 isolates ofRhizoctonia spp. obtained from 95 soil samples collected from different fields in the USA, 42 (27.5%) isolates were hypovirulent or non-pathogenic on cabbage (tested on tap water agar plus 250 μg/ml chloramphenicol plates). Of these, 14 (33.3% of the np-R) isolates protected >60% of the cabbage seedlings againstR. solani, and the best eight isolates protected 73–95% of the cucumber seedlings. The np-R isolates RU56-8 (AG-P) and RU89-1 [AG-B(o)] induced the highest resistance against hypocotyl challenge inoculation with virulentR. solani (38.3–85.7%), whereas most of the challenged control seedlings (85–100%) collapsed. Similarly, isolates RU56-8 and RU89-1 induced the highest resistance (22.2–87.5%) against hypocotyl challenge inoculation withPythium aphanidermatum, whereas most of the challenged control seedlings collapsed (90–100%). Isolates RU56-8 and RU89-1 significantly reduced the lesion numbers and area/leaf (to 8.9–42.0% of the control) caused by challenge inoculation of the first true leaves withPseudomonas syringae pv.lachrymans. No np-R isolate could be recovered from the upper hypocotyls or from the leaves, indicating that there was no contact between the inducer and the pathogen. Root colonization with some np-R increased seedling tolerance to low soil moisture levels.     

Survey of Bacterial and Fungal Seedborne Diseases in Imported and Domestic Potato Seed Tubers

Potato seed tubers are imported to Israel from northern Europe and planted in spring; tubers harvested early from the spring crop are used as seed for the autumn crop. Although only seed lots registered as certified are imported, a previous survey (1984–1994) indicated that most imported lots were affected by latent or active infections caused byErwinia carotovora,Streptomyces scabies, Rhizoctonia solani, Fusarium spp. andSpongospora subterranae. The survey was extended until 1998, and included additional pathogens:Ralstonia solanacearum,Helminthosporium solani, Colletotrichum coccodes andVerticillium dahliae. Most of these pathogens were also monitored in domestic seed tubers, and are reported for the first time. Brown rot was not observed in any of the imported lots. Blackleg and soft rot caused byErwinia spp. were detected in most of the imported lots; however, less than 7% of the lots were contaminated at high levels, while approximately 65% were contaminated at moderate levels. Common scab was detected in most of the imported lots; 51% of the imported lots were contaminated at moderate or high levels, whereas only 6.5% of the domestic seed lots were contaminated at these levels. Black scurf was detected in most of the imported lots; on average, 47.3%, 44.2% and 1.4% of the lots were contaminated at low, moderate and high levels, respectively, and only 7.1% were disease-free. In contrast, most of the domestic lots were either disease-free (45.4%) or had a low disease incidence (37.3%). Only 16.7% of the lots were moderately infected and 0.2% were highly contaminated. Silver scurf was observed in most of the imported lots during all years of the survey, with no differences among the producing countries; on average, 22.7%, 66.1% and 7.5% of the lots were contaminated at low, moderate and high levels, respectively, and only 3.7% were disease-free. Most of the domestic lots (76%) were disease-free and only 6.6% were infected at moderate or high levels. Black dot was observed in a considerable portion of the shipments from Holland during all years of the survey, particularly in 1998, when 34% of the lots were infected. The shipments from France and Germany were infected at low levels, except in 1998, when 19% and 11% of the lots, respectively, arrived infected. In shipments from Scotland and Ireland low incidences of the disease were observed in 1994 and 1995. In the domestic lots, black dot incidence was low (<2.4%) except in 1996, when 11% of the lots were infected.V. dahliae was monitored only in domestic seed tubers. The incidence of disease-free lots was 56–64%, whereas in 20–30% of the lots the level of infection was <5%, and in 6–16% of the lots the level was >5%. The survey findings demonstrate transmission of seedborne pathogens; most of these pathogens can become established in the soil and eventually cause severe outbreaks of disease in potatoes grown in Israel. http://www.phytoparasitica.org posting May 16, 1999.     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.     

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl⁻¹) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.     

Weed hosts ofVerticillium dahliae in crete: Susceptibility, symptomatology and significance

A survey of common and uncommon weed species usually showing Verticillium wilt symptoms was carried out during 1992–2000 in Crete, Greece.Verticillium dahliae was isolated in 48 out of 182 sampled fields, in which several weed species were grown, from several locations in Oropedio, Lasithi. Altogether, 124 isolates ofV. dahliae were recovered from the vascular stem-tissue of 19 weed species, belonging to ten botanical families. Pathogenicity trials with 13 out of 19 weed species that have never been reported as hosts of the fungus, using for inoculation isolates which originated from the same weed species, resulted in infection of all of them, showing various disease symptoms. Seven weed species (Anthemis melanolepis, Cardaria draba, Convolvulus arvensis, Erodium sp.,Euphorbia helioscopia, Helminthotheca echioides andSinapis alba) are new hosts worldwide, and six additional species (Euphorbia sp.,Lactuca serriola, Raphanus raphanistrum, Sinapis arvensis, Sonchus oleraceus andTrifolium sp.) are new hosts for Greece. The most susceptible (isolation frequency: 27.9–52.8%, moderate disease severity) species were:Capsella bursa-pastoris, C. draba, Chenopodium album, Senecio vulgaris andSolanum nigrum. Less susceptible (isolation frequency: 4.8–17.8%, slight disease severity) were:Amaranthus sp.,A. melanolepis, C. arvensis, Erodium sp.,Euphorbia sp.,E. helioscopia, H. echioides, L. serriola, Malva sylvestris, R. raphanistrum, S. alba, S. arvensis, S. oleraceus andTrifolium sp. Some species —C. draba, C. album, L. serriola andS. nigrum L. — that usually showed external and vascular wilt symptoms, occasionally exhibited only reduced growth. Visible symptoms under natural field conditions in all 13 weed species that had never been reported as hosts ofV. dahliae were similar to those observed after their artificial inoculation. The fungus was not isolated fromFoeniculum vulgare ssp.piperitum, Oxalis corniculata andStellaria media, among other species. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and peroxidise

Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.     

PCR-based determination of colonization patterns during potato tuber infection by single and multiple pathogens

Potato tubers piled in storage are prone to infection by numerous pathogens. Each pathogen can cause damage alone, but severe losses often arise when more than one pathogen is involved. Currently, only a visual diagnosis is practiced on potato tubers before storing them, which does not allow any prediction of further disease spread. The aim of the present study was to determine differences in patterns of tissue colonization by several tuber decay pathogens and how late blight infection affects further tuber colonization by other important tuber pathogens. This study was conducted using artificial inoculation of potato tubers and PCR to provide an early and accurate diagnosis of disease development for major potato tuber rots, and to assess potential synergism/antagonism between Phytophthora infestans and other pathogens in stored tubers. In order to accurately follow the progress of each pathogen in tuber tissues, samples were collected over time from both the surface (peel, 0–2 mm depth) and internal tissues (flesh, depth > 2 mm) of the tubers at various distances from the inoculation site, at 3, 5, 7, 10, 12, 14, 17, and 19 days after inoculation. Successful detection of single or multiple pathogens was achieved using specific PCR-primers for each pathogen. Pathogens were always detected several centimeters ahead of the visible lesions. This tracking enabled us to determine the extent of colonization both on the tuber’s surface and in internal tissues by each tested pathogen, either after single or multiple infections involving P. infestans as the primary pathogen. The presence of P. infestans was shown to enhance the development of Pectobacterium atrosepticum and to slow down that of P. erythrospetica and Pythium ultimum. No noticeable effect on further tuber colonization by F. sambucinum, V. dahliae or V. albo-atrum was observed in the presence of P. infestans. This approach involving more than one pathogen is more realistic than classical studies considering single pathogens, and may be helpful in monitoring the sanitary status of stored tubers. Our results make the outcome of certain combinations of pathogens in potato tubers more predictable and may result in more efficient preventive measures.     

Characterization of a phytotoxic glycoprotein produced byPhoma eupyrena — A pathogen on water lettuce

Phoma eupyrena, the causal agent of leaf blight disease of water lettuce, when purified by affinity and ion exchange chromatography produced an extracellular glycoprotein (Pe 65) in concentrations of ∼ 8 μg ml⁻¹ in the stationary culture. Coomassie-blue stained SDS-PAGE analysis of culture filtrates and purified Pe 65 showed its molecular mass to be 65 kDa. The blighting and necrosis of leaf tissues were observed within 4–6 days when 1–5 μg of Pe 65 was injected into the mesophyll of water lettuce. These symptoms closely resembled those caused by foliar inoculation with the pathogen. Recognition of Pe 65 by N-glycosidase F treatment and by polyclonal antibodies raised in rabbit against the whole glycoprotein, indicated that the protein is a highly glycosylated protein (50% carbohydrate) and that it is strongly enclosed by the antigenic glycosidic moiety. http://www.phytoparasitica.org posting May 6, 2003.     

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (10³ and 10⁴ conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (10⁵ and 10⁶ conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.     

Dry mycelium of<Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> induces resistance against verticillium wilt and enhances growth of cotton plants

Dry mycelium (DM) ofPenicillium chrysogenum and its water extract (DME) were examined for their effects on induced resistance against Verticillium wilt and plant growth of cotton in the greenhouse. Soil application of 0.1–5% DM or 0.5–5% DME provided significant protection against the wilt, relative to the control. As neither DM nor DME inhibited mycelial growth ofVerticillium dahliae in vitro, it is suggested that the disease-controlling effects of DM or DME are attributed to induced resistance. DME (5%), as well as DME treated with chloroform or cold acetone, were as effective as 2% DM in reducing disease severity of Verticillium wilt, implying that the resistance-inducing substance(s) in DM are mostly water-soluble, with neither proteins nor lipids likely to be responsible for the induction of resistance. No significant difference in root colonization withV. dahliae was found between control-inoculated and 2% DM- or 5% DME-inoculated plants. However, colonization of hypocotyls and epicotyls was drastically suppressed by either 2% DM or 5% DME relative to the control. Treatments with 2% DM or 5% DME significantly increased ionically-bound peroxidase (POX) activity in roots, hypocotyls and the second leaf of cotton plants, with the hypocotyls expressing the highest increase. Soil application of DM or DME increased plant height, fresh and dry weight of inoculated and non-inoculated cotton plants, relative to their corresponding controls. It is concluded that DM may be used in cotton crops to promote plant growth and to induce resistance againstV. dahliae. POX might be associated with the defense against Verticillium wilt. http://www.phytoparasitica.org posting Jan. 9, 2002.