Peter V. Oudemans

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control

ABSTRACT A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.

Habitat and host indicate lineage identity in Colletotrichum gloeosporioides s.l. from wild and agricultural landscapes in North America.

Understanding the factors that drive the evolution of pathogenic fungi is central to revealing the mechanisms of virulence and host preference, as well as developing effective disease control measures. Prerequisite to these pursuits is the accurate delimitation of species boundaries. Colletotrichum gloeosporioides s.l. is a species complex of plant pathogens and endophytic fungi for which reliable species recognition has only recently become possible through a multi-locus phylogenetic approach. By adopting an intensive regional sampling strategy encompassing multiple hosts within and beyond agricultural zones associated with cranberry (Vaccinium macrocarpon Aiton), we have integrated North America strains of Colletotrichum gloeosporioides s.l. from these habitats into a broader phylogenetic framework. We delimit species on the basis of genealogical concordance phylogenetic species recognition (GCPSR) and quantitatively assess the monophyly of delimited species at each of four nuclear loci and in the combined data set with the genealogical sorting index (gsi). Our analysis resolved two principal lineages within the species complex. Strains isolated from cranberry and sympatric host plants are distributed across both of these lineages and belong to seven distinct species or terminal clades. Strains isolated from V. macrocarpon in commercial cranberry beds belong to four species, three of which are described here as new. Another species, C. rhexiae Ellis & Everh., is epitypified. Intensive regional sampling has revealed a combination of factors, including the host species from which a strain has been isolated, the host organ of origin, and the habitat of the host species, as useful indicators of species identity in the sampled regions. We have identified three broadly distributed temperate species, C. fructivorum, C. rhexiae, and C. nupharicola, that could be useful for understanding the microevolutionary forces that may lead to species divergence in this important complex of endophytes and plant pathogens.

Phenology of Apothecium Production in Populations of Monilinia vaccinii-corymbosi from Early- and Late-Maturing Blueberry Cultivars.

ABSTRACT Pseudosclerotia were evaluated for differences in timing of apothecium development in four controlled experiments conducted over a 2-year period. In a separate experiment, conidia from 10 randomly selected isolates from both of the fungal populations were used to inoculate open flowers. Germination of pseudosclerotia produced from these artificial inoculations also was evaluated. The timing and rate of shoot elongation for cvs. Weymouth and Jersey were assessed in one greenhouse and two field experiments. Average development times for the fungal population from cv. Weymouth were 8 to 15 days earlier or 33 to 42% less than those for the population from cv. Jersey. The fungal population from Weymouth also exhibited less variation in development times for each developmental stage measured. Similarly, germination of pseudosclerotia produced in artificial inoculations differed between populations. On average, pseudosclerotia derived from the Weymouth population produced apothecia 16 days earlier. During spring 1995 and 1996, vegetative and truss buds on cv. Weymouth developed 4 to 16 days earlier than those on cv. Jersey. These results demonstrate that M. vaccinii-corymbosi exhibits variation in timing of pseudosclerotia germination and apothecium development within and between populations. We hypothesize that differences observed in the timing of apothecium development are related to the fitness of the populations on their original host cultivars and were selected by host phenology.

Estimation of spatial and spectral properties of phytophthora root rot and its effects on cranberry yield

Abstract Current agricultural practices are aimed at maximizing productivity while minimizing the area of cultivated land. This is especially important in cranberry production because strict federal guidelines curtail development of new cranberry acreage on wetlands. A major component of this research is focused on the chronic effects of phytophthora root rot (PRR) because of the difficulties in detection and the significant impact on yields. PRR causes a reduction in root mass, which results in reduced canopy biomass and alters the spectral reflectance characteristics of the canopy. Detection of acute cases of PRR using color-infrared (CIR) aerial photography is straightforward from apparent bare soil on May images; however, the level of detectable chronic infection is unknown. The objectives of this study are to investigate the relationships between soil characteristics, spectral properties of the crop surface, and the severity of Phytophthora effects on cranberries. Soil, pathogen, and crop data were entered in a GIS and the relationships among the factors were studied using geostatistical methods and surface maps of the relevant GIS layers. These maps were then compared and incorporated with the data derived from remotely sensed images (CIR aerial photographs—May, 2001 and July, 2001). The spatial pattern of stressed vegetation was fairly consistent through 5 years and corresponded to spread of PRR chronic injury and low yield. The disease develops in surface depressions with low infiltration rates, which have high soil water content during July–August. The results suggest that early-season (May) CIR images have more predictive power for the yield and vine density, whereas late-season (July) images are more correlated with PRR and soil infiltration rate.

Variation and Heritability of Phenology in the Fungus Monilinia vaccinii-corymbosi on Blueberry.

ABSTRACT The germination of field-collected pseudosclerotia and the development of apothecia from eight New Jersey populations of the mummy berry fungus Monilinia vaccinii-corymbosi were evaluated under controlled conditions in the greenhouse. Development data for apothecia were used to describe the timing of apothecium formation and to estimate broad- and narrow-sense heritabilities of fungal phenology. Mean development times for the formation of apothecia ranged from 35.4 to 54.7 days. The mean development times for populations collected from early-season cv. Weymouth ranged from 35.4 to 39.6 days and were significantly shorter than the development times for three of the four populations collected from late-season cv. Jersey (46.9 to 54.7 days) or for the population collected from mixed stands of cultivated blueberries (42.7 days). The development of populations from late cultivars planted in very close proximity to early cv. Weymouth was early (36.5 to 39.0 days) and not significantly different from the development of populations collected from cv. Weymouth. Phenotypic and genetic variances of apothecium development for individual populations ranged from 18.9 to 44.8 and 7.2 to 30.9, respectively. Broad-sense heritabilities of apothecia development for each fungal population, calculated by partitioning phenotypic variation into genetic and environmental components, ranged from 0.31 to 0.78. Narrow-sense heritabilities of apothecia development, based on parent-offspring regression, ranged from 0.58 to 0.78. These results indicate that populations of M. vaccinii-corymbosi differ in phenology and that a significant portion of the phenological variation within populations is genetic. Thus, it is plausible to propose that the phenology of apothecium development is a component of fungal fitness and that host phenology can influence the timing of pathogen development.

A long terminal repeat retrotransposon Cgret from the phytopathogenic fungus Colletotrichum gloeosporioides on cranberry.

Abstract A repetitive DNA element cloned from the cranberry fruit rot pathogen Colletotrichum gloeosporioides has been characterized. Sequence data indicate that it is a long terminal repeat (LTR) retrotransposon of 7,916 base pairs. LTR of 544 base pairs occur at either end of an internal region of 6,828 base pairs. This element, designated Cgret (C. gloeosporioides retrotransposon), encodes two putative polypeptides which have high homology to the gag and pol genes of other fungal retrotransposons. The sequence and structure suggest that Cgret is a member of the gypsy group of LTR retrotransposons. The Cgret retrotransposon was present in all of the cranberry isolates of the fungus C. gloeosporioides from New Jersey and Massachusetts, but not in the cranberry isolates from Wisconsin or Chile. Polymorphisms were detected among field isolates of C. gloeosporioides from various hosts, using hybridization probes derived from the LTR and the reverse transcriptase domain of Cgret. The structural integrity of Cgret suggests that it is still a functional retrotransposon and may be used as a molecular marker to study the genetic diversity distribution of this fungal pathogen.

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.

Identification of a New Phytophthora Species Causing Root and Runner Rot of Cranberry in New Jersey

ABSTRACT In New Jersey, Phytophthora cinnamomi is the pathogen most commonly isolated from diseased roots and runners of the cultivated cranberry (Vaccinium macrocarpon). A second distinct species of Phytophthora has been isolated from dying cranberry plants and surface irrigation water. This species is homothallic with paragynous antheridia and ellipsoid-limoniform, nonpapillate sporangia. It was tentatively identified as P. megasperma in an earlier report. Laboratory experiments demonstrate that the cardinal temperatures for vegetative growth are between 5 and 30 degrees C with an optimum near 25 degrees C. Sporangia are produced at temperatures between 10 and 20 degrees C with the majority of sporangia produced at 10 and 15 degrees C. In pathogenicity tests, no growth effect was observed on cranberry plants (cv. Early Black) when tests were conducted at 25 degrees C; however, significant reductions in plant growth occurred when tests were conducted at 15 degrees C. This species was insensitive to metalaxyl but was sensitive to buffered phosphorous acid. Sequence analysis of the internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2 regions place these isolates in Phytophthora clade 6 with greatest similarity to Phytophthora taxon raspberry. To our knowledge, this is the first report of isolates of this affiliation in North America. However, the observation of low temperature preferences makes this species unique in an otherwise high temperature clade. The isolates described in this study are tentatively classified as Phytophthora taxon cranberry.

Investigating the Potential of Area-to-Area and Area-to-Point Kriging for Defining Management Zones for Precision Farming of Cranberries

Cranberries are harvested by flooding the field and agitating vines so the fruit, which float can be skimmed from the surface and loaded into barrels. This harvesting method makes application of standard precision farming practices difficult. This paper investigates the potential of combining Area-to-Area (AtoA) and Area-to-Point (AtoP) kriging of yield totals from individual fields with remotely sensed data for defining within-field management zones.

Host Resistance to Monilinia vaccinii-corymbosi in Flowers and Fruits of Highbush Blueberry

Monilinia vaccinii-corymbosi, the causal agent of mummy berry disease, infects blueberry flowers via the gynoecial pathway. To describe the expression of host resistance in highbush blueberry (Vaccinium corymbosum), fungal growth in the styles and colonization of the locules were compared among five blueberry cultivars in a series of controlled greenhouse experiments. Styles were harvested 1 and 4 days postinoculation, and the length colonized by hyphae was determined using fluorescence microscopy. At 8 weeks after inoculation, fruit were harvested and scored for the presence of hyphae in the locules. The infection frequency of styles ranged from 0.33 to 0.71, and only cv. Weymouth had significantly lower infection frequency than the other cultivars. The mean length of the colonized portion of the stylar canal ranged from 0.126 to 0.434 mm after 1 day and 1.62 to 3.59 mm after 4 days. Hyphae in the styles of cv. Weymouth exhibited the least growth, whereas hyphae in the styles of cultivars Jersey and Rancocas were significantly longer. The distance of style penetrated for cultivars Bluecrop and Coville was intermediate. The mean disease incidence of locules differed significantly. Values for cultivars Weymouth and Jersey were the smallest (0.038 and 0.039) and largest (0.249 and 0.236), respectively. The results demonstrate that a component of resistance to infection by M. vaccinii-corymbosi is expressed during growth in the gynoecial pathway.