Odile Carisse

Cytological aspects of compost-mediated induced resistance against fusarium crown and root rot in tomato.

ABSTRACT The potential of a pulp and paper mill residues compost for the control of crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici was ultrastructurally investigated. Peat moss amended with compost substantially reduced disease-associated symptoms. Addition of Pythium oligandrum to either peat moss alone or peat moss amended with compost resulted in a considerable reduction in disease incidence compared with controls grown in peat moss alone. Histological and cytological observations of root samples from Fusarium-inoculated plants revealed that the beneficial effect of compost in reducing disease symptoms is associated with increased plant resistance to fungal colonization. One of the most prominent facets of compost-mediated induced resistance concerned the formation of physical barriers at sites of attempted fungal penetration. These structures, likely laid down to prevent pathogen ingress toward the vascular elements, included callose-enriched wall appositions and osmiophilic deposits around the sites of potential pathogen ingress. Invading hyphae, coated by the osmiophilic material, showed marked cellular disorganization. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin was altered in severely damaged hyphae. A substantial increase in the extent and magnitude of the cellular changes induced by compost was observed when P. oligandrum was supplied to the potting substrate. This finding corroborates the current concept that amendment of composts with specific antagonists may be a valuable option for amplifying their beneficial properties in terms of plant disease suppression.

Effect of Fall Application of Fungal Antagonists on Spring Ascospore Production of the Apple Scab Pathogen, Venturia inaequalis.

ABSTRACT The influences of Microsphaeropsis sp., M. arundinis, Ophiostoma sp., Diplodia sp., and Trichoderma sp., all antagonists of Venturia inaequalis, on ascospore production were evaluated under natural conditions and compared with urea and Athelia bombacina, a known antagonist. In the autumn, the fungi were applied to leaf disks artificially inoculated with V. inaequalis and to scabbed apple (Malus domestica) leaves incubated under controlled and natural conditions. In addition, large-scale trials were conducted with Microsphaeropsis sp. applied either as a foliar postharvest spray or as a ground application at 90% leaf fall. All fungal isolates, except Ophiostoma sp., were recovered from the leaf material that overwintered in the orchard. All treatments, except those with Ophiostoma sp., resulted in a significant reduction in V. inaequalis ascospore production on the leaf disks incubated under controlled conditions or in the orchard. In 1997, leaves with apple scab lesions treated with urea or Microsphaeropsis sp. produced significantly fewer ascospores of V. inaequalis than did nontreated leaves, with a reduction of 73.0 and 76.3%, respectively. In 1998, leaves treated with Microsphaeropsis sp., urea, Trichoderma sp., A. bombacina, and M. arundinis reduced ascospore production by 84.3, 96.6, 75.2, 96.6, and 52.2%, respectively. Based on all tests combined, the most efficient isolate was Microsphaeropsis sp. Postharvest applications of Microsphaeropsis sp. reduced the total amount of airborne ascospores trapped by 70.7 and 79.8% as compared with the nontreated plots in 1997 and 1998, respectively. Microsphaeropsis sp. provided a significant and consistent reduction in ascospore production in all tests.

Effect of Microsphaeropsis sp. on the Production of Perithecia and Ascospores of Gibberella zeae

The potential of Microsphaeropsis sp. (isolate P130A) as an antagonist of Gibberella zeae was tested under in vitro and field conditions. Firstly, an in vitro method of ascospore production was developed on wheat and corn residues. The plant type (corn or wheat), residue type (straw/stalk or grain), and incubation conditions (closed or open) had a significant effect on ascospore production. Perithecia were more abundant on wheat and corn grain incubated under open conditions. On these two substrates, the application of Microsphaeropsis sp. significantly reduced ascospore production. On wheat, the antagonist had a significant effect when applied 2 weeks before (-2), at the same time (0), and 4 weeks after (+4) inoculation with G. zeae, with 1.73, 0.31, 1.11, and 1.36 log ascospores per cm2 for the control, -2, 0, and +4 weeks treatments, respectively. On corn, Microsphaeropsis sp. had a significant effect when applied 2 weeks before, at the same time, 4 weeks after, and 6 weeks after inoculation with G. zeae with 3.02, 0.23, 1.29, 2.35, and 2.22 log ascospores per cm2 for the control, -2, 0, +4, and +6 weeks treatments, respectively. When applied to crop residues in the field as postharvest or preplanting applications, Microsphaeropsis sp. had no effect on the pattern of perithecia maturation, but significantly reduced the number of perithecia produced on two sampling dates, May 1998 and July 1999. There is a potential to biologically reduce the initial inoculum of G. zeae; however, more work is needed to optimize the efficacy of the biocontrol agent.

Spatiotemporal Relationships Between Disease Development and Airborne Inoculum in Unmanaged and Managed Botrytis Leaf Blight Epidemics

Comparatively little quantitative information is available on both the spatial and temporal relationships that develop between airborne inoculum and disease intensity during the course of aerially spread epidemics. Botrytis leaf blight and Botrytis squamosa airborne inoculum were analyzed over space and time during 2 years (2002 and 2004) in a nonprotected experimental field, using a 6 x 8 lattice of quadrats of 10 x 10 m each. A similar experiment was conducted in 2004 and 2006 in a commercial field managed for Botrytis leaf blight using a 5 x 5 lattice of quadrats of 25 x 25 m each. Each quadrat was monitored weekly for lesion density (LD) and aerial conidium concentration (ACC). The adjustment of the Taylors power law showed that heterogeneity in both LD and ACC generally increased with increasing mean. Unmanaged epidemics were characterized in either year, with aggregation indices derived from SADIE (Spatial Analysis by Distance Indices). For LD, the aggregation indices suggested a random pattern of disease early in the season, followed by an aggregated pattern in the second part of the epidemic. The index of aggregation for ACC in 2002 was significantly greater than 1 at only one date, while it was significantly greater than 1 at most sampling dates in 2004. In both years and for both variables, positive trends in partial autocorrelation were observed mainly for a spatial lag of 1. In 2002, the overall pattern of partial autocorrelations over sampling dates was similar for LD and ACC with no significant partial autocorrelation during the first part of the epidemic, followed by a period with significant positive autocorrelation, and again no autocorrelation on the last three sampling dates. In 2004, there was no significant positive autocorrelation for LD at most sampling dates while for ACC, there was a fluctuation between significant and non-significant positive correlation over sampling dates. There was a significant spatial correlation between ACC at given date (t(i)) and LD 1 week later (t(i + 1)) on most sampling dates in both 2002 and 2004 for the unmanaged and managed sites. It was concluded that LD and ACC were not aggregated in the early stage of epidemics, when both disease intensity and airborne conidia concentration were low. This was supported by the analysis of LD and ACC from a commercial field, where managed levels of disease were low, and where no aggregation of both variables was detected. It was further concluded that a reliable monitoring of airborne inoculum for management of Botrytis leaf blight is achievable in managed fields using few spore samplers per field.

Selection of biological agents from composts for control of damping-off of cucumber caused by Pythium ultimum

The microflora of three composts were studied to isolate and test microorganisms for biological control of Pythium ultimum on greenhouse-grown cucumber. A more diverse bacterial population was observed in compost from paper mill sludge (170 groups) than in composts from plant waste and from manure (75 and 88 groups, respectively). In compost from paper mill sludge, 12 fungal species were isolated, compared with 22 and 26 in composts from plant waste and from manure, respectively. Selected bacterial and fungal isolates were evaluated in assays on agar plates against P. ultimum. A total of 10 bacterial isolates and 4 fungal isolates significantly (P ≤ 0.0001) reduced P. ultimum growth rate. These isolates were evaluated in the greenhouse for control of damping-off of cucumber. Disease incidence and severity and foliage and root masses were measured 3 weeks after soil inoculation with the microbial agents and P. ultimum. Under greenhouse conditions, Zygorrhinchus moelleri and Bacillus marinus were the most effective microorganisms against damping-off, followed by Penicillium thomii, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Graphium putredinis. These isolates were tested in a second greenhouse trial and the best control was achieved with Z. moelleri and Penicillium thomii.

Spatial Distribution of Venturia inaequalis Airborne Ascospores in Orchards.

ABSTRACT Apple scab (Venturia inaequalis) causes important economic losses in many apple production areas of the world. The disease is controlled by numerous fungicide applications regardless of the presence of ascospores in the orchard. Airborne ascospore concentration (AAC) can be measured in real time to time fungicide applications. However, the level of heterogeneity of the AAC in commercial orchards was unknown. Consequently, the spatial distribution of V. inaequalis ascospores was studied in a commercial apple orchard of 0.43 ha. The potential ascospore dose (PAD) and AAC were measured in 40 quadrats each of 108 m(2). In each quadrat, the AAC was monitored during the major rain events in spring 1999 and 2000 using spore samplers. The variance-to-mean ratio for the PAD and for most of the AAC sampling dates was >1, indicating an aggregated pattern of distribution. None of the frequency distributions of the most important ascospore ejection events followed the Poisson probability distribution, indicating that the pattern of distribution was not random. For all events, AAC had an aggregated pattern of distribution as suggested by the negative binomial distribution. The PAD followed neither the Poisson nor the negative binomial distribution. Geostatistical analyses confirmed the aggregated pattern of distribution. The cultivars had an effect on the PAD and AAC distribution pattern, but both PAD and AAC were not uniformly distributed within a block of the same cultivar. Therefore, the number, location, and height of samplers required to estimate AAC in orchards need to be investigated before using information on AAC for decision making.

Cytochemical Investigation of the Antagonistic Interaction Between a Microsphaeropsis sp. (Isolate P130A) and Venturia inaequalis.

ABSTRACT In an attempt to better understand the mode of action of the antagonistic fungus Microsphaeropsis sp., the interaction between this fungus and Venturia inaequalis was studied, using both light and electron microscopy. Cytological observations indicated that the antagonistic interaction between the two fungi likely involves a sequence of events, including (i) attachment and local penetration of Microsphaeropsis sp. into V. inaequalis hyphae; (ii) induction of host structural response at sites of potential antagonist entry; (iii) alteration of host cytoplasm; and (iv) active multiplication of antagonistic cells in pathogen hyphae, leading to host cell breakdown and release of the antagonist. The interaction was investigated further by gold cytochemistry. The use of gold-complexed beta-1,4-exoglucanase and wheat germ agglutinin/ovomucoid-gold complex to localize cellulosic beta-1,4-glucans and chitin monomers, respectively, resulted in regular labeling of V. inaequalis cell walls. This finding supports other studies refuting the classification of ascomycetes as only a glucan-chitin group. At an advanced state of parasitism, the labeling pattern of cellulose and chitin, which clearly showed that the level of integrity of these compounds was affected, suggested the production of cellulolytic and chitinolytic enzymes by Microsphaeropsis sp. Wall appositions formed in V. inaequalis in response to the antagonists attack contained both cellulose and chitin. However, penetration of this newly formed material frequently succeeded. This study provides the first detailed picture of the cytological events associated with mycoparasitism in V. inaequalis.

Sensitivity of Botrytis squamosa to Different Classes of Fungicides

An automated quantitative (AQ) assay was compared with radial growth on solid media and with dry weight in liquid culture for assaying fungicide sensitivity in Botrytis squamosa, the causal agent of onion leaf blight. Five isolates of B. squamosa were assayed for sensitivity to mancozeb (Dithane DG) and iprodione (Rovral) at five concentrations (0.5, 1.0, 5.0, 10.0, and 50 ppm). For mancozeb, the correlations between 50% effective concentration (EC50) values obtained with the three assays were not significant; however for iprodione, correlations between EC50 values for AQ and radial growth and for AQ and dry weight were significant (r = 0.98 and 0.99, respectively). The AQ method was less time consuming and more reliable than the two standard assays. The AQ method was used to evaluate the sensitivity of 35 field isolates of B. squamosa to mancozeb (Dithane DG), iprodione (Rovral), vinclozolin (Ronilan DF), and chlorothalonil (Bravo 500). All isolates were sensitive to mancozeb (EC50 ranged from 3.36 to 12.97) and chlorothalonil (EC50 < 1.5 μg/ml), but four isolates were insensitive to both iprodione (EC50 ≥ 3.98 μg/ml) and vinclozolin (EC50 ≥ 17.49 μg/ml). The ratio of the EC50 values of the least-sensitive and the most-sensitive isolates of B. squamosa was 1.08, 3.86, 6.98, and 37.59 for chlorothalonil, mancozeb, iprodione, and vinclozolin, respectively. Cross-resistance was observed for the two dicarboximide fungicides, iprodione and vinclozolin, with a significant correlation (r = 0.94) in the sensitivity of the 35 isolates to these two fungicides.

Effect of Microsphaeropsis sp. Strain P130A on Germination and Production of Sclerotia of Rhizoctonia solani and Interaction Between the Antagonist and the Pathogen

Microsphaeropsis sp. strain P130A was evaluated for the control of tuber-borne inoculum of Rhizoctonia solani based on the viability of sclerotia produced in vitro and on both the viability and production of tuber-borne sclerotia. The interactions between the antagonist and the pathogen, as well as the effect of the toxins produced by the antagonist on mycelial growth of R. solani were studied using transmission electron microscopy. On sclerotia produced in vitro, for all incubation periods (1 to 42 days), Microsphaeropsis sp. significantly reduced germination. Percent germination of sclerotia treated with Microsphaeropsis sp. decreased with increasing incubation period from an average of 82.0% after 1 day to stabilize at an average of 5.8% after 35 days. Similarly, percent germination of tuber-borne sclerotia was significantly lower when tubers were treated with Microsphaeropsis sp. Both 2% formaldehyde and Microsphaeropsis sp. treatments significantly reduced sclerotia germination to approximately 10% after 42 days of incubation at 4 degrees C. Furthermore, on tubers treated with the antagonist, the number of sclerotia per square centimeter decreased from 1.6 to 0.5 during the 8 months of storage at 4 degrees C, whereas an increase from 1.2 to 7.8 sclerotia per square centimeter was observed on untreated tubers. Microsphaeropsis sp. (strain P130A) colonized hyphae of R. solani within 4 days after contact on culture media. Transmission electron microscopic observations showed that the antagonist induced a rupture of the pathogen plasma membrane and that a chitin-enriched matrix was deposited at sites of potential antagonist penetration. Host penetration was not associated with pathogen cell wall alterations, which occurred at the time of progress of the antagonist in the pathogen cytoplasm. In the presence of a crude extract of Microsphaeropsis sp., cells of R. solani showed cytoplasm disorganization and breakdown of plasma membranes. Antibiosis and mycoparasitism were involved in the antagonism of R. solani by Microsphaeropsis sp., but the sequence by which these events occur, as well as the significance of wall appositions produced by R. solani, is yet to be established.

In vitro evaluation of fungal isolates for their ability to influence leaf rheology, production of pseudothecia, and ascospores of Venturia inaequalis

As part of an ongoing research project on biological control of apple scab, this study presents a novel approach for the in vitro selection of potential antagonists of the saprophytic phase of Venturia inaequalis. A collection of forty-two fungal isolates were tested for their in vitro ability to degrade apple leaf tissue, inhibit pseudothecia, and ascospore production. The inhibition of ascospore production cannot always be linked reliably with leaf degradation or the evaluation of pseudothecia production. Consequently, ascospore production was retained as the most useful screening parameter. Six isolates proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of fungal and bacterial saprophytes can now be based on a reliable and simple in vitro screening method.