Annemiek C. Schilder

Efficacy of chlorine dioxide gas sachets for enhancing the microbiological quality and safety of blueberries.

In response to increasingly stringent microbial specifications being imposed by purchasers of frozen blueberries, chlorine dioxide (ClO2) gas generated by a dry chemical sachet was assessed for inactivation of Listeria monocytogenes, Salmonella spp., and Escherichia coli O157:H7 as well as five yeasts and molds known for blueberry spoilage. Fresh blueberry samples (100 g) were separately inoculated with cocktails of L. monocytogenes, Salmonella, E. coli O157:H7 (three strains each), or yeasts and molds (five strains each) to contain approximately 10(6) CFU/g and exposed to ClO2 (4 mg/liter, 0.16 mg/g) for 12 h in a sealed 20-liter container (99.9% relative humidity) at approximately 22 degrees C. After gassing, 25 g of blueberries was added to 225 ml of neutralizing buffer, pulsified for 1 min, and plated using standard procedures to quantify survivors. This treatment yielded reductions of 3.94, 3.62, 4.25, 3.10, and 3.17 log CFU/g for L. monocytogenes, Salmonella, E. coli O157:H7, yeasts, and molds, respectively. Thereafter, 30 lugs of uninoculated blueberries (approximately 9.1 kg per lug) were stacked on 1.2 by 1.2-m pallets (5 lugs per level x six levels), tarped, and exposed to ClO2 (18 mg/liter, 0.13 mg/g) for 12 h. After gassing, significant (P < 0.05) reductions of 2.33, 1.47, 0.52, 1.63, and 0.48 log CFU/g were seen for mesophilic aerobic bacteria, coliforms, E. coli, yeasts, and molds, respectively, compared with non-gassed controls. No significant differences (P > 0.05) in microbial inactivation were seen between lug levels and, with one exception (mesophilic aerobic bacteria), between the bottom and top surface of individual lugs. Based on these findings, ClO2 sachets may provide a simple, economical, and effective means of enhancing the microbial shelf life and safety of blueberries.

Phylogenetic and experimental evidence for host-specialized cryptic species in a biotrophic oomycete.

Assortative mating resulting from host plant specialization has been proposed to facilitate rapid ecological divergence in biotrophic plant pathogens. Downy mildews, a major group of biotrophic oomycetes, are prime candidates for testing speciation by host plant specialization. Here, we combined a phylogenetic and morphological approach with cross-pathogenicity tests to investigate host plant specialization and host range expansion in grapevine downy mildew. This destructive disease is caused by Plasmopara viticola, an oomycete endemic to North America on wild species and cultivated grapevines. Multiple genealogies and sporangia morphology provide evidence that P. viticola is a complex of four cryptic species, each associated with different host plants. Cross-inoculation experiments showed complete host plant specialization on Parthenocissus quinquefolia and on Vitis riparia, whereas cryptic species found on V. aestivalis, V. labrusca and V. vinifera were revealed to be less specific. We reconstructed the recent host range expansion of P. viticola from wild to cultivated grapevines, and showed that it was accompanied by an increase in aggressiveness of the pathogen. This case study on grapevine downy mildew illustrates how biotrophic plant pathogens can diversify by host plant specialization and emerge in agrosystems by shifting to cultivated hosts. These results might have important implications for viticulture, including breeding for resistance and disease management.

A First Assessment of the Cranberry Fruit Rot Complex in Michigan

Samples of ripe fruit were taken at harvest from all eight commercial cranberry farms in Michigan over a 3-year period to determine the distribution and incidence of fruit rot diseases and the fungal pathogens associated with rotted fruit. Totals of 23, 33, and 28 beds were sampled in 1999, 2000, and 2001, respectively. Fruit rot incidence varied widely among beds and farms and ranged from 5 to 97% (mean 33.4%) in 1999, 1 to 91% (mean 26.3%) in 2000, and 1 to 67% (mean 12.8%) in 2001. Differences in fruit rot incidence were observed among cultivars, but rankings differed among farms. In general, cultivars Ben Lear, Bergman, and Pilgrim tended to have lower and Beckwith and WSU61 higher fruit rot incidence than other cultivars grown in the same location. Colletotrichum acutatum, Pestalotia vaccinii, and Phyllosticta vaccinii were the fungi most frequently recovered from rotted fruit. Fusicoccum putrefaciens, Phomopsis vaccinii, Physalospora vaccinii, Allantophomopsis lycopodina, Coleophoma empetri, and Botrytis cinerea were isolated occasionally in 1999. The isolation frequency of Physalospora vaccinii, Phomopsis vaccinii, and C. empetri increased markedly in 2000. Glomerella cingulata was first detected in 2001. Fusicoccum putrefaciens was most common in the northern and Glomerella cingulata in the southern growing areas. A comparison of sound and rotted fruit from selected beds showed that Phyllosticta elongata predominated in sound fruit, whereas G. cingulata predominated in rotted fruit.

Strobilurin (QoI) Resistance in Populations of Erysiphe necator on Grapes in Michigan

Powdery mildew, caused by Erysiphe necator, is the most common and destructive disease of grapes (Vitis spp.) worldwide. In Michigan, it is primarily controlled with fungicides, including strobilurins (quinone outside inhibitors [QoIs]). Within the United States, resistance to this class of fungicides has been reported in E. necator populations in some east coast states. Among 12 E. necator isolates collected from five Michigan vineyards in 2008, one carried the G143A single-nucleotide mutation responsible for QoI resistance. This isolate was confirmed to be resistant in a conidium germination assay on water agar amended with trifloxystrobin at 0.001, 0.01, 0.1, 1, 10, or 100 μg/ml and salicylhydroxamic acid (100 mg/liter). The mutant isolate was able to germinate on media amended with 100 μg/ml trifloxystrobin, whereas a representative wild-type isolate did not germinate at concentrations higher than 0.1 μg/ml. In 2009, 172 isolates were collected from a total of 21 vineyards (juice and wine grapes): three vineyards with no fungicide application history (baseline sites), six research vineyards, and 12 commercial vineyards. QoI resistance was defined as the effective concentration that inhibited 50% of conidial germination (EC50) > 1 μg/ml. Isolates from baseline sites had EC50 values mostly below 0.01 μg/ml, while isolates that were highly resistant to trifloxystrobin (EC50 > 100 μg/ml) occurred in five research and three commercial wine grape vineyards at frequencies of 40 to 100% and 25 to 75% of the isolates, respectively. The G143A mutation was detected in every isolate with an EC50 > 1 μg/ml. These results suggest that fungicide resistance may play a role in suboptimal control of powdery mildew observed in some Michigan vineyards and emphasizes the need for continued fungicide resistance management.

Root Colonization by Ericoid Mycorrhizae and Dark Septate Endophytes in Organic and Conventional Blueberry Fields in Michigan

Ericoid mycorrhizae are interfaces for nutrient exchange between specialized fungi and roots of blueberries and other ericaceous plants. Dark septate endophytes also associate with roots of the Ericaceae and many other plant families, but the nature of the interaction is not well understood. We measured ericoid mycorrhizae and dark septate endophyte colonization in organic and conventional blueberry fields matched by soil series and, if possible, cultivar and field age. The percentage of hair root cells with ericoid mycorrhizae was generally higher in organic than conventional fields, while dark septate endophyte colonization was higher in sand than muck soils. Across both muck and sandy soil types, ericoid mycorrhizal colonization was negatively correlated with field age, and dark septate endophyte colonization was negatively correlated with soil carbon, total soil nitrogen, and soil ammonium levels. When only sandy soils were included in the analysis, ericoid mycorrhizal colonization was positively correlated with soil pH and hair-root diameter, and negatively correlated with field age, soil ammonium levels, and total soil nitrogen. On sandy soils, dark septate endophyte colonization was not correlated with any measured variable. The functions of ericoid mycorrhizae and dark septate endophytes in commercial blueberry fields require further investigation.

Geographic distribution of cryptic species of Plasmopara viticola causing downy mildew on wild and cultivated grape in eastern North America.

The putative center of origin of Plasmopara viticola, the causal agent of grape downy mildew, is eastern North America, where it has been described on several members of the family Vitaceae (e.g., Vitis spp., Parthenocissus spp., and Ampelopsis spp.). We have completed the first large-scale sampling of P. viticola isolates across a range of wild and cultivated host species distributed throughout the above region. Sequencing results of four partial genes indicated the presence of a new P. viticola species on Vitis vulpina in Virginia, adding to the four cryptic species of P. viticola recently recorded. The phylogenetic analysis also indicated that the P. viticola species found on Parthenocissus quinquefolia in North America is identical to Plasmopara muralis in Europe. The geographic distribution and host range of five pathogen species was determined through analysis of the internal transcribed spacer polymorphism of 896 isolates of P. viticola. Among three P. viticola species found on cultivated grape, one was restricted to Vitis interspecific hybrids within the northern part of eastern North America. A second species was recovered from V. vinifera and V. labrusca, and was distributed across most of the sampled region. A third species, although less abundant, was distributed across a larger geographical range, including the southern part of eastern North America. P. viticola clade aestivalis predominated (83% of isolates) in vineyards of the European winegrape V. vinifera within the sampled area, indicating that a single pathogen species may represent the primary threat to the European host species within eastern North America.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

Status Update on Organic Blueberries in Michigan

The Great Lakes Region is an important region of blueberry production in the U.S., producing 30% of the annual U.S. production. In Michigan, blueberry acreage increased from 17,724 acres on 590 farms in 2002 to 21,758 acres on 840 farms in 2007. However, despite considerable market potential for organic blueberries, less than 1% of total Michigan blueberry acreage is organically certified. There is high interest and demand for organic blueberries from the Great Lakes Region, and the Michigan State University Blueberry Team has been working on organic production methods over the past 4 years. The establishment of the Michigan State University Organic Blueberry Research and Extension Planting on the Michigan State University campus has been one of the major accomplishments of the Michigan State Uuniversity research team. The objective of this project has been to study practices associated with soil health, nutrition, disease, and insect and weed control. Additional organic blueberry projects in Michigan have focused on studying the interaction of blueberry mulches and compost on nutrient release, and on-station and on-farm testing of OMRI-approved pesticides.

Floral Scent Mimicry and Vector-Pathogen Associations in a Pseudoflower-Inducing Plant Pathogen System

Several fungal plant pathogens induce ‘pseudoflowers’ on their hosts to facilitate insect-mediated transmission of gametes and spores. When spores must be transmitted to host flowers to complete the fungal life cycle, we predict that pseudoflowers should evolve traits that mimic flowers and attract the most effective vectors in the flower-visiting community. We quantified insect visitation to flowers, healthy leaves and leaves infected with Monilinia vaccinii-corymbosi (Mvc), the causative agent of mummy berry disease of blueberry. We developed a nested PCR assay for detecting Mvc spores on bees, flies and other potential insect vectors. We also collected volatiles from blueberry flowers, healthy leaves and leaves infected with Mvc, and experimentally manipulated specific pathogen-induced volatiles to assess attractiveness to potential vectors. Bees and flies accounted for the majority of contacts with flowers, leaves infected with Mvc and healthy leaves. Flowers were contacted most often, while there was no difference between bee or fly contacts with healthy and infected leaves. While bees contacted flowers more often than flies, flies contacted infected leaves more often than bees. Bees were more likely to have Mvc spores on their bodies than flies, suggesting that bees may be more effective vectors than flies for transmitting Mvc spores to flowers. Leaves infected with Mvc had volatile profiles distinct from healthy leaves but similar to flowers. Two volatiles produced by flowers and infected leaves, cinnamyl alcohol and cinnamic aldehyde, were attractive to bees, while no volatiles manipulated were attractive to flies or any other insects. These results suggest that Mvc infection of leaves induces mimicry of floral volatiles, and that transmission occurs primarily via bees, which had the highest likelihood of carrying Mvc spores and visited flowers most frequently.

Genome sequence and detection of peach rosette mosaic virus

Peach rosette mosaic disease was first described in the 1940s affecting peach and plum. It was later determined that peach rosette mosaic virus (PRMV) is the causal agent of the disease. PRMV, a member of the genus Nepovirus, infects several perennial crops including stone fruit, grape and blueberry as well as several weed species found in orchards around the world. The molecular characterization of the virus is limited to partial genome sequences making it difficult to develop reliable and sensitive molecular detection tests; the reason that detection is routinely performed using ELISA with antibodies risen against a single virus isolate. Given the potential economic impact of the virus and the modes of transmission which, in addition to nematodes, include seed we studied PRMV in more depth using a modified dsRNA extraction protocol to obtain the virus genome. We determined the full nucleotide sequence and developed a protocol that detects conserved regions present in RNA 1 and RNA 2, making it an excellent alternative to the detection protocols used today.