Cinerea Spores Research Articles

HEALTH MONITORING OF PLANTS BY THEIR EMITTED VOLATILES: A TEMPORARY INCREASE IN THE CONCENTRATION OF METHYL SALICYLATE AFTER PATHOGEN INOCULATION OF TOMATO PLANTS AT GREENHOUSE SCALE

This paper describes a method to alert growers of the presence of a pathogen infection in their greenhouse based on the detection of pathogen-induced emissions of volatile organic compounds (VOCs) from plants. Greenhouse-grown plants were inoculated with spores of a fungus to learn more about this concept. The specific objective of the present study was to determine whether VOCs are detectable after inoculation, and if so, to determine the time course of the concentrations of these compounds. To achieve this objective, we inoculated 60 greenhouse-grown tomato plants (Lycopersicon esculentum) with an aqueous suspension of Botrytis cinerea spores. Upon inoculation, the greenhouse air was sampled semi-continuously with a one hour time interval until 72 hours after inoculation (HAI). The samples were transferred to the laboratory and analysed using gas chromatography - mass spectrometry. Ten leaves were randomly selected to monitor the visible symptoms of infection. The severity of these visual symptoms was assessed at 0, 24, 48, and 72 HAI. Results demonstrated no detection of C6-compounds, and an almost constant concentration of all monoterpenes, most sesquiterpenes, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. However, the concentration of methyl salicylate increased 10-fold and 3-fold at 32 and 34 HAI respectively. At 24 HAI, 10% of the selected leaves showed mild symptoms while 20% of the selected leaves showed mild symptoms at 48 HAI. These results indicate that methyl salicylate might alert a grower of the presence of a B. cinerea infection of tomato plants at greenhouse scale. Further research is required to confirm these findings.

Potencial para o biocontrole de Botrytis cinerea por leveduras em sistema integrado de cultivo de lírio

O objetivo deste trabalho foi comparar uma população de leveduras do filoplano de lírio de um sistema convencional de cultivo com outra de sistema integrado, além de avaliar o antagonismo da levedura Sporidiobolus pararoseus a Botrytis cinerea. A comunidade de leveduras foi estimada pelo método da queda dos balistoporos nos terços inferior, médio e superior das plantas, bem como nas faces adaxial e abaxial das folhas. Nos discos de folhas de plantas do sistema integrado, foi alta a presença da população de leveduras, tendo sido maior na face adaxial das folhas do terço médio das plantas. Para avaliação do antagonismo, foram aplicadas aos discos de folhas as concentrações de 10(5), 10(6) e 10(7) células mL-1 da levedura, em três períodos de inoculação: 24 horas antes; simultânea a; e 24 horas após a inoculação de 10(4) esporos mL-1 de B. cinerea. A incidência, a percentagem de área de discos colonizados e a esporulação de B. cinerea foram avaliadas por meio de diagrama de notas, a partir do quarto dia após a inoculação. Todas as concentrações da levedura reduziram a esporulação de B. cinerea nos discos de folha, em comparação à testemunha, e a concentração de 10(7) esporos mL-1 foi a mais eficiente.

Potential use of hydroxytyrosol-rich extract from olive mill wastewater as a biological fungicide against Botrytis cinerea in tomato

Olive mill wastewater (OMW) is a rich source of antifungal agents and has a merit as alternative to synthetic fungicides. To develop disease control agents against Botrytis cinerea, a hydroxytyrosol-rich (29.27% weight:dry weight) olive mill wastewater (HROMW) and a hydroxytyrosol-rich (52.67% weight:dry weight) extract (HRE) were prepared from fresh OMW using hydrolysis and post-hydrolysis purification processes. The HROMW and HRE showed strong fungicidal activity against B. cinerea with minimal fungicidal concentrations (MFCs) of 14.36–28.72 and 28.72–57.44 mg l−1 (dry weight), respectively. According to EN 1275: 1997 standard method, HROMW and HRE at 1.25% (dry weight:volume) showed powerful disinfectant properties resulting in a reduction in the viability of B. cinerea spores by >4 log units after 30 min of contact time. The HROMW or HRE foliar spray treatments, of the B. cinerea inoculated tomato plants, resulted in disease control percentage of 95 and 91%, respectively, whereas 70% was conferred by benomyl treatment. Although, HRE possessed a similar disease severity reduction than HROMW, it showed great advantage by fortifying plants and by improving their growth and healthy aspect. Indeed, HRE-treated plants showed significantly better: (i) plant height (146.2 and 130.5%), (ii) average stem diameter (135% and 110%), (iii) leaf number (163.6 and 128.5%), and (iv) leaf colour (chlorophyll a content) (144.4 and 103%) than benomyl-treated and non-treated plants, respectively. This study revealed that HRE offers a natural and effective extract to control grey mould.

The use of aerobiological methods for forecasting Botrytis spore concentrations in a vineyard

Grey mould, caused by Botrytis cinerea, is one of the most common diseases in Galician vineyards. Knowledge of local airborne spore concentrations is of great value for developing models to predict fungal propagule concentrations, thus enabling phytosanitary treatments to be applied when a real risk of infection is detected. In order to develop an accurate model for forecasting airborne B. cinerea spore concentrations, a phenological and aerobiological survey was carried out in a vineyard located in Galicia (north-west Spain), from 2004 to 2008. Phenological observations were made on 20 vines of each of three grape varieties (Treixadura, Godello and Loureira) using the BBCH standardised phenological scale. A Hirst-type volumetric spore-trap was used for the aerobiological monitoring. The study sought to chart Botrytis spore concentrations as function of grapevine phenological stage. The highest spore concentrations over the grapevine cycle as a whole were recorded in 2008 (37299 spores), and the lowest in 2005 (1700 spores). In the five study years, the highest concentrations were registered during stage 8 (berry ripening), stage 7 (fruit development) and stage 5 (inflorescence emergence). The weather-related parameters displaying the most significant correlation with spore concentrations were dew point and relative humidity. ARIMA (Autoregressive Integrated Model of Running Mean) time-series models was used to forecast daily spore concentrations, considering B. cinerea spore concentrations and weather data as predictor variables.

Using combinations of biocontrol agents to control Botrytis cinerea on strawberry leaves under fluctuating temperatures

Experiments were conducted with Botrytis cinerea on strawberry leaves to investigate where combinations of commercially available biological control agents (BCAs) might control B. cinerea more effectively than individual BCAs. Specifically, we studied the persistence of biocontrol activities, spread of BCAs among leaves, and biocontrol efficacy in relation to application regimes: mixed versus single BCA, pre-versus post-inoculation application, and sequential versus simultaneous application. Three BCA products (Sentinel, Serenade and Trianum) were used for this study. Overall, Serenade did not significantly reduce sporulation of B. cinerea on strawberry leaf discs whereas Sentinel and Trianum gave a similar and significant biocontrol efficacy. Biocontrol efficacy remained almost unchanged 10 days after application at 20/20°C (day/night) or 24/16°C temperature regimes. In contrast, reduced biocontrol efficacy at 26/14°C suggests BCA survival was reduced under these conditions. Incidence of B. cinerea sporulation on leaf discs was ca. 60% higher on leaves that emerged after the BCA application than on leaves directly exposed to BCA, indicating insufficient amount of the BCA had managed to spread to new leaves. Combinations of BCAs, whether applied simultaneously or sequentially (48 h apart), did not improve disease control over the most effective BCA within the combination applied alone. This indicated possible antagonism or interference between the BCAs. Results suggested that there was significant antagonism for most combinations of the three BCAs tested and the degree of antagonism increased as the time from BCA application to pathogen introduction lengthened.

Release of lipoxygenase products and monoterpenes by tomato plants as an indicator of Botrytis cinerea‐induced stress

Changes in emission of volatile organic compounds (VOCs) from tomato induced by the fungus Botrytis cinerea were studied in plants inoculated by spraying with suspensions containing B. cinerea spores. VOC emissions were analysed using on-line gas chromatography-mass spectrometry, with a time resolution of about 1 h, for up to 2 days after spraying. Four phases were delimited according to the starting point and the applied day/night rhythm of the experiments. These phases were used to demonstrate changes in VOC flux caused by B. cinerea infestation. Tomato plants inoculated with B. cinerea emitted a different number and amount of VOCs after inoculation compared to control plants that had been sprayed with a suspension without B. cinerea spores. The changes in emissions were dependent on time after inoculation as well as on the severity of infection. The predominant VOCs emitted after inoculation were volatile products from the lipoxygenase pathway (LOX products). The increased emission of LOX products proved to be a strong indicator of a stress response, indicating that VOC emissions can be used to detect plant stress at an early stage. Besides emission of LOX products, there were also increases in monoterpene emissions. However, neither increased emission of LOX products nor of monoterpenes is specific for B. cinerea attack. The emission of LOX products is also induced by other stresses, and increased emission of monoterpenes seems to be the result of mechanical damage induced by secondary stress impacts on leaves.

Enhancing disease resistance in peach fruit with methyl jasmonate

AbstractBACKGROUND: Peaches are susceptible to microbial decay during postharvest distribution at ambient temperature. To search for effective alternatives to currently used fungicides for disease control, in this study the effect of methyl jasmonate (MeJA) on disease resistance and fruit decay of peaches after harvest in response to pathogen attack was investigated.RESULTS: Freshly harvested peaches were treated with 1 µmol L−1 MeJA vapour at 20 °C for 24 h. At 0, 12, 24 and 36 h after this treatment, both treated and untreated fruits were artificially wounded and inoculated with Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer spore suspension (1 × 105 spores mL−1) and then incubated at 20 °C for 6 days. MeJA treatment significantly reduced the postharvest diseases. Incubation for 12 h was the optimal length of time after MeJA treatment, resulting in the lowest disease incidence and lesion diameter for all pathogens. The activities of defence enzymes including chitinase, β‐1,3‐glucanase, phenylalanine ammonia‐lyase, polyphenol oxidase and peroxidase were enhanced by MeJA treatment, and the level of total phenolics in MeJA‐treated fruit was also higher than that in control fruit. In addition, MeJA affected hydrogen peroxide (H2O2)‐metabolising enzymes such as superoxide dismutase, catalase and ascorbate peroxidase and induced a higher level of H2O2 during incubation, which might serve as a signal to induce resistance against P. expansum.CONCLUSION: MeJA was effective in reducing decay and might enhance disease resistance in peach fruit by increasing levels of antipathogenic proteins and antimicrobial phenolic compounds. Copyright © 2009 Society of Chemical Industry

Targeted selection of antagonistic microorganisms for control ofBotrytis cinereaof strawberry in New Zealand

A range of microorganisms, with previously identified antagonistic properties towards a variety of plant pathogens, were screened for their biological control activity against the foliar necrotroph Botrytis cinerea. On detached, senescing strawberry leaves the most successful isolate, Trichoderma atroviride LU132, was able to inhibit B. cinerea lesion development and significantly reduce sporulation compared to the control treatment. T. atroviride LU132 was further evaluated under field conditions and, over a 2-year period, on three different strawberry cultivars, was able to significantly suppress B. cinerea sporulation on leaves and flower parts giving a similar level of disease control as the fungicide fenhexamid. The mechanisms of action of T. atroviride LU132were postulated to be a combination of competition for sugars, production of non-volatile compounds and possible mycoparasitism. The strategic approach described in this paper demonstrates an efficient way of screening microorganisms for biocontrol activity and validates the notion that certain microorganisms can be antagonistic to a variety of plant pathogens.

Exo‐β‐1,3‐Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

AbstractYeast exo‐β‐1,3‐glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 103 to 80 × 103 spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 103 to 80 × 103, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5‐day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β‐glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea, respectively, could be considered.

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10 8 CFU ml −1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10 5 spores ml −1; While the blue mold decay was completely inhibited at concentrations of 5 × 10 8 CFU ml −1, at challenged with P. expansum spores suspension of 5 × 10 4 spores ml −1 . These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10 5 CFU per wound to 2.24 × 10 7 CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.

Control of postharvest pear diseases using Rhodotorula glutinis and its effects on postharvest quality parameters

Rhodotorula glutinis was evaluated for its activity in reducing postharvest gray mold decay and blue mold decay of pear caused by Botrytis cinerea and Penicillium expansum respectively, and in reducing natural decay development of pear fruits, as well as its effects on postharvest quality of fruits. There was a significant negative correlation between concentrations of the yeast cells and infectivity of the pathogens. At concentrations of R. glutinis at 5 × 108 CFU/ml, the gray mold decay was completely inhibited after 7 days incubation at 20 °C, while the control fruit had 100% disease incidence and 2.15 cm lesion diameter respectively, at challenged with B. cinerea spores suspension of 1 × 105 spores/ml; No completely control was got to blue mold, when pear fruits stored at 20 °C for 7 d (challenged with P. expansum spores suspension of 5 × 104 spores/ml), but the decay was distinctly prevented with 20% and 0.60 cm of disease incidence and lesion diameter respectively, while the control fruits were 100% and 2.74 cm, respectively. Rapid colonization of the yeast in wounds was observed during the first 1 d at 20 °C, and then the populations stabilized for the remaining storage period. On pear wounds kept at 4 °C, rapid colonization of the yeast in wounds was observed during the first 3 d, and then the increase in population density of R. glutinis turned slow, which continued over 6 d after application of the antagonist until it reached a high level. Then, the populations stabilized for the remaining storage period. In the test on PDA plates, R. glutinis significantly inhibit the growth of B. cinerea and P. expansum. Spore germination of pathogens in PDB was greatly controlled in the present of living yeast cell suspensions. R. glutinis significantly reduced the natural development of decay of pear following storage at 20 °C for 7 days or at 4 °C for 30 days followed by 20 °C for 5 days, and did not impair quality parameters, including mass loss, firmness, TSS, ascorbic acid or titratable acidity. Thus, the application of R. glutinis can be an alternative to chemicals for control of postharvest diseases on pear fruits.

Botrytis cinerea spore balance of a greenhouse rose crop

Fungal pathogens are among the most virulent bioagressors of protected crops. For sustainable plant production and to protect the crop against these airborne organisms one must determine their origin, i.e. whether they come from outside the greenhouse or are produced inside it. We considered Botrytis cinerea spore concentration as a particular physical species which is transported by air in the same way as heat, CO 2, water vapour or any tracer gas. We constructed a mass balance of the viable Botrytis spores on the whole-greenhouse volume to assess the inside production of spores and their exchanges. The different elements in the spore balance are considered and are determined experimentally: • spore transfer into or out of the greenhouse is deduced from the difference between the inside and outside spore concentrations, measured by spore traps, multiplied by the whole-greenhouse ventilation rate; • spore deposition on soil and crop and spore impaction on the plants is deduced from observations made using Petri dishes; • inside spore production is the differential term deduced from the whole-greenhouse volume balance. The first measurements and calculations have shown that the presence of insect-proof nets across the greenhouse openings strongly influences the spore balance, as the nets intercept the B. cinerea spores proportionally to the nets’ solidity. Based on wind tunnel experiments, the interception capacity of the nets was quantified and the whole model completed in consequence. These results show the diurnal Botrytis spore balance over 3–4-week periods in the autumn and spring season for a young rose plantation. In agreement with visual observations of B. cinerea sporulation within the greenhouse, it is shown that the origin of the inocula is predominantly internal (2/3 in quantity). However, due to outside climate and greenhouse opening, it can vary widely from one period to another, with a tendency to become predominantly internal as the crop ages.

The Necrotroph Botrytis cinerea Induces a Non-Host Type II Resistance Mechanism in Pinus pinaster Suspension-Cultured Cells

Models of non-host resistance have failed to account for the pathogenicity of necrotrophic agents. During the interaction of Pinus pinaster (maritime pine) with the non-host necrotrophic pathogen Botrytis cinerea, the generation and scavenging of reactive oxygen species (ROS) and the induction of the hypersensitive response (HR) were analyzed. Elicitation of maritime pine suspended cells with B. cinerea spores resulted in the biphasic induction of ROS. The phase I oxidative burst was dependent on calcium influx, while the phase II oxidative burst also depended on NADPH oxidase, protein kinase activity, and de novo transcription and protein synthesis. A decline was observed in catalase (CAT) and superoxide dismutase (SOD) activity, together with the down-regulation of Fe-Sod1, chlCu, Zn-Sod1 and csApx1, suggesting a coordinated response towards a decrease in the ROS-scavenging capacity of maritime pine cells during challenge. Following the second oxidative burst, programmed cell death events characteristic of the HR were observed. The results suggest the ROS-mediated and cell-breach-independent activation of Type II non-host resistance during the P. pinaster-B. cinerea interaction.

Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens

BackgroundVascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.ResultsB. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX.ConclusionB. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this type of cell death in vascular plants, our results suggest that E.c. carotovora CFSCC1 containing HrpN and B. cinerea could also induce this type of cell death in Physcomitrella. Our studies thus establish Physcomitrella as an experimental host for investigation of plant-pathogen interactions and B. cinerea and elicitors of E.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant.

Influence of application time on the establishment, survival, and ability of Clonostachys rosea to suppress Botrytis cinerea sporulation on rose debris

The influence of application time and the length of exposure to sunlight on survival, establishment, and ability of Clonostachys rosea to suppress Botrytis cinerea sporulation on rose debris were investigated in a climate-controlled greenhouse (experiment 1) and in a plastic-covered greenhouse (experiment 2). C. rosea conidia germinated significantly greater on treatments kept out of direct light for all application times. However, the germination incidence was negatively correlated with the application time for all treatments, independent of sunlight exposure. The recovery of viable conidia from leaves and the tissues colonized by C. rosea reduced exponentially as exposure to sunlight increased (from 0 to 8 h). The relative humidity (RH) was positively correlated with germination independent of sunlight exposure ( r=0.95 and r=0.97, for shadow and sun, respectively). On treatments exposed to sunlight, the global radiation was negatively correlated with germination, especially during the 3 h following inoculation ( r=−0.94). Despite the effects of exposure to sunlight on C. rosea, the suppression of B. cinerea sporulation was only marginally affected (suppression of 94.5–100% and 65–93%, respectively, for experiment 1 and experiment 2). These results show the ability of C. rosea to withstand adverse environmental conditions and still provide suppression of B. cinerea sporulation on rose debris.

Mutations in LACS2, a Long-Chain Acyl-Coenzyme A Synthetase, Enhance Susceptibility to Avirulent Pseudomonas syringae But Confer Resistance to Botrytis cinerea in Arabidopsis

We identified an Arabidopsis (Arabidopsis thaliana) mutant, sma4 (symptoms to multiple avr genotypes4), that displays severe disease symptoms when inoculated with avirulent strains of Pseudomonas syringae pv tomato, although bacterial growth is only moderately enhanced compared to wild-type plants. The sma4 mutant showed a normal susceptible phenotype to the biotrophic fungal pathogen Erysiphe cichoracearum. Significantly, the sma4 mutant was highly resistant to a necrotrophic fungal pathogen, Botrytis cinerea. Germination of B. cinerea spores on sma4 mutant leaves was inhibited, and penetration by those that did germinate was rare. The sma4 mutant also showed several pleiotropic phenotypes, including increased sensitivity to lower humidity and salt stress. Isolation of SMA4 by positional cloning revealed that it encodes LACS2, a member of the long-chain acyl-CoA synthetases. LACS2 has previously been shown to be involved in cutin biosynthesis. We therefore tested three additional cutin-defective mutants for resistance to B. cinerea: att1 (for aberrant induction of type three genes), bodyguard, and lacerata. All three displayed an enhanced resistance to B. cinerea. Our results indicate that plant cutin or cuticle structure may play a crucial role in tolerance to biotic and abiotic stress and in the pathogenesis of B. cinerea.

EXPRESSION PROFILES OF POTATO PROTEINASE INHIBITORS POTENTIALLY USEFUL FOR THE CONTROL OF PHYTOPATHOGENIC FUNGI

Proteinase inhibitors (PIs) are small ubiquitous proteins showing a variety of biological functions in plants, such as stabilization of proteins, modulation of apoptosis and defence against pathogens. They are particularly abundant in storage organs, such as seeds and tubers, and can accumulate in other plant tissues following a variety of different stimuli. Besides patatin, the major protein constituents of potato (Solanum tuberosum) tubers are low molecular weight PIs, such as protease inhibitors I (PI-1), II (Pin2) and Kunitz-type (PKPIs). PI-1 and PKPI inhibitors are coded in potato plants by relatively small gene families and synthesized as pre-pro-proteins (signal peptide-propeptidemature proteins). A minimum of 21 Kunitz inhibitor genes (subdivided, on the basis of their sequence homology, into group A, B, and C) has been found to be present in the potato genome, while contrasting data have been reported for the number of genes belonging to the PI-1 family. In addition, PKPI and PI-1 accumulate in different plant tissues in response to biotic or abiotic stresses. However, little information is today available about the regulation (e.g. after nematode infection) of individual PI-1 genes or PKPI groups. In the present work, a PI mixture, obtained from potato (cv.. Desiree) sprouts and showing a strong inhibitory activity against Botrytis cinerea fungal proteases, spore germination, hyphal elongation and necrotic activity, was purified and sequenced. Five novel full length PI-1 cDNAs (PPI3B2, PPI3A2, PPI3A4, PPI2C4 and PPI2C1A) and two PKPI genes (PKI1 and PKI2) were isolated, sequenced and characterized. Southern analysis, performed to confirm the presence of PKI1 and PPI3B2 genes and to determine the number of PI-1 genes in the genome, indicated that the PKI1 gene is present as a single copy in the highly polymorphic family of the Kunitz inhibitors of the cultivar Desiree. On the other hand, the hybridizing bands obtained with the PPI3B2 probe (at least nine) indicate that this gene could be part of a small multigene family (~ 10 members). Alignment of cloned and database retrieved PI inhibitor genes was used to design family, group or gene specific primers to individually analyze target transcripts by semiquantitative RT-PCR. Expression profiles of the three PKPI groups (A, B and C), Pin2 and PR1 (pathogenesi related 1) gene families, and the PI-1 family genes PPI3A2, PPI3B2, PPI2C4 were studied in various tissues of Desiree, LB7/4/c-I-7 and P40 potato genotypes, after wounding and infection by Globodera rostochiensis. PI, but not PR1 genes, were found to be involved in the plant response triggered by the nematode G. rostochiensis, indicating an activation of the defense pathway via jasmonic acid. Moreover, individual PI-1 genes and PKPI groups were expressed in a tissue and genotype specific manner after biotic and abiotic stresses. The differences in the PI expression patterns concerned the intensity, type of inhibitors and rapidity of induction.

Management of understorey to reduce the primary inoculum of Botrytis cinerea: Enhancing ecosystem services in vineyards

Botrytis bunch rot, caused by Botrytis cinerea (Pers.: Fr) is an important disease of grapevines that causes worldwide crop losses and reductions in wine quality. The pathogen predominantly overwinters on vine debris on the vineyard floor. In the current work, organic mulches were used to enhance biological degradation of vine debris to reduce levels of B. cinerea primary inoculum the following season. Four mulch types (anaerobically and aerobically fermented marc (grape pressings), inter-row grass clippings, and shredded office paper) were applied under 10-year-old Riesling vines in a 10-replicate randomized block design in New Zealand over two consecutive years. Plastic mesh bags, each containing naturally infected vine debris, were placed under vines on bare ground (control) and at the soil-mulch interface, in winter (July) 2003 and 2004. In each year, half the bags were recovered at flowering (December) and the remainder at leaf plucking (February), for assessment of B. cinerea sporulation from the vine debris and debris degradation rate. Bait lamina probes, which measure soil biological activity, were placed in the soil-mulch interface three weeks before each of the two bag-recovery dates in both years and were then removed and assessed at the same times as were the bags. All mulches led to a reduction in B. cinerea sporulation. This reduction was significantly correlated with elevated rates of vine debris decomposition and increased soil biological activity. Over both years, compared with the controls, all treatments gave a 3- to 20-fold reduction in B. cinerea sporulation, a 1.6- to 2.6-fold increase in vine debris degradation and in the two marc and the paper treatments, a 1.8- to 4-fold increase in activity of soil organisms. These results show the potential of enhanced soil organism activity in the soil for disrupting the B. cinerea life cycle. The implications of these results for infection levels of grapes are currently being investigated.

Biological control of postharvest spoilage caused by Penicillium expansum and Botrytis cinerea in apple by using the bacterium Rahnella aquatilis

The epiphytic bacterium Rahnella aquatilis, isolated from fruit and leaves of apples, was tested for antagonistic properties against Penicillium expansum and Botrytis cinerea on Red Delicious apple fruit. In “in vitro” assays, this bacterium inhibited completely the germination of P. expansum and B. cinerea spores, but it needed direct contact with the spores to do it. However the putative mechanism seemed be different for the two pathogens. The bacterium did not produce extracellular antibiotic substances and when the acute toxicity test was performed no mortality, toxicity symptoms or organ alterations of the test animals (Wistar rats) were observed. Assays of biological control of P. expansum and B. cinerea on apple fruit were carried out at different temperatures. At 15 °C and 90% RH, the incidence of disease caused by P. expansum on apples stored for 20 days, was reduced by nearly 100% by R. aquatilis (10 6 cells/ml), while in the case of B. cinerea, the reduction of decay severity was nearly 64% but there was no reduction in the incidence of disease. At 4 °C and 90% RH the treatment with the bacterium significantly inhibited the development of B. cinerea on apples stored for 40 days and the incidence of disease was reduced by nearly 100%, while the incidence of disease caused by P. expansum at 4 °C was 60%. The results obtained show that R. aquatilis would be an interesting microorganism to be used as a biocontrol agent.

Postharvest ethanol and potassium sorbate treatments of table grapes to control gray mold

Germination of Botrytis cinerea spores on potato dextrose agar after a 30 s immersion in 10 or 20% ethanol was 87 and 56%, respectively, compared to 99% among untreated controls. After similar immersion in 0.5 or 1.0% potassium sorbate, 84 and 68% of the spores germinated, respectively. Addition of 0.5 and 1.0% potassium sorbate to 10 and 20% ethanol solution significantly increased the inhibition of spore germination. The germination of spores after 30 s immersion in 20% ethanol plus 0.5% potassium sorbate was 9.7%. The incidence of gray mold, caused by B. cinerea, on detached berries of ‘Flame Seedless’ grapes immersed for 30 s in water, 10 and 20% ethanol, and 0.5 or 1.0% potassium sorbate was 55.2, 42.1, 31.0, 37.7, or 24.4%, respectively. Addition of 0.5 and 1.0% potassium sorbate to 10 and 20% ethanol reduced decay to 10% or less and was more effective than either alone. After 30 days of storage at 1 °C, the combination of 20% ethanol either with 0.5 or 1.0% potassium sorbate was equal in efficacy to commercial SO 2 generator pads in reducing the incidence of gray mold on ‘Thompson Seedless’ grapes. None of the combinations of ethanol and potassium sorbate injured the berries.