Maryna Serdani

Reconstructing the early evolution of Fungi using a six-gene phylogeny

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

Description of Cryptosporiopsis kienholzii and species profiles of Neofabraea in major pome fruit growing districts in the Pacific Northwest USA.

The objectives of this study were i) to give a taxonomic description of a fungus phylogenetically related to Neofabraea and assign the name Cryptosporiopsis kienholzii to this fungus, ii) to expand previous Neofabraea species profiles from infected apple and pear fruit collected from major pome fruit production districts in Oregon and Washington, and iii) to determine the sensitivity of Neofabraea alba, Neofabraea malicorticis, Neofabraea perennans, and C. kienholzii to a range of fungicides. A name is given herein to the anamorph of the fungus previously called Neofabraea sp. nov. in the literature, and the fungus is designated as C. kienholzii. The teleomorph of this fungus has never been observed in vivo or in vitro. N. alba, N. perennans, and C. kienholzii accounted for 61.3, 35.6, and 3.1 %, respectively, of 608 isolates obtained from pear fruit and 6.0, 81.3, and 12.7 % of 150 isolates from apple fruit. N. alba was the most common species in Oregon and N. perennans was most common in Washington. N. malicorticis was not found in any of the districts and may be limited to the wet climates west of the Cascade mountain range. C. kienholzii occurs in pome fruit orchards from southern Oregon to North Central Washington, and this is the first report of C. kienholzii in the state of Washington. The four most effective fungicides for control of bulls-eye rot caused by all species were thiabendazole, thiophanate-methyl, pyrimethanil, and pyraclostrobin+boscalid. Other fungicides gave acceptable control of some species of Neofabraea but not others. There was good agreement of the microbioassay with fungicide evaluations using wound-inoculated pear fruit for five of seven fungicides, but the microbioassay was not consistent enough for determination of the sensitivity of Neofabraea species to new fungicides.

Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.

Bacterial Canker of Sweet Cherry in Oregon—Infection of Horticultural and Natural Wounds, and Resistance of Cultivar and Rootstock Combinations

This study was done to (i) compare seven types of natural or horticultural injuries and wounds for incidence, severity, and mortality of infection of sweet cherry (Prunus avium) by Pseudomonas syringae pv. syringae; (ii) determine the relative resistance to bacterial canker of 14 cultivar-rootstock combinations; (iii) determine if P. syringae pv. syringae is transmitted by contaminated pruning tools; and (iv) determine if summer and winter pruning cuts become resistant to infection. Infection occurred at all of the seven types of injury and wound sites on both cvs. Sunset Bing and Golden Heart. Infection of inoculated wounds made in spring and summer (heading cuts when trees were planted, scoring cuts, and summer pruning) resulted in the greatest canker incidence and severity. Inoculation of heading cuts resulted in the highest tree mortality (86%). Bing and Sweetheart were the most susceptible cultivars while Regina and Rainier appeared to be more resistant. Bing trees had the highest mortality of any cultivar with 70% dead at the end of the 3-year study. Canker severity of the three rootstocks varied considerably but mortality was greatest for trees on Gisela 6 (77%). Bacterial canker was not transmitted in summer or winter by cutting through active cankers, then immediately using the same pruning tool to make heading cuts on healthy trees. Heading cuts became resistant to infection after about 1 week in summer and 3 weeks in winter. Results are discussed as part of an integrated management program for bacterial canker of sweet cherry.

Methodology for Determining Relationships Between Inoculum Concentration of Botrytis cinerea and Penicillium expansum and Stem End Decay of Pear Fruit

The objective of this research was to determine quantitative relationships between incidence of stem end decay of pear fruit and inoculum concentration of Botrytis cinerea and Penicillium expansum using dry conidia applied to pear fruit in a settling tower. Five concentrations of conidia were applied to pear fruit, fruit were stored at -1°C for 8 months, and stem end decay was evaluated. In addition, conidia were washed from the surface of inoculated fruit, and DNA was extracted and quantified with real-time polymerase chain reaction (PCR). The linear regression relationships between percent stem end gray mold and B. cinerea conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01). At the highest inoculum dose introduced into the settling tower, conidia per liter of air, conidia per square centimeter, and percent stem end gray mold at 8 months after inoculation were 12, 31, and 39, respectively for 2000 and 6, 33, and 67, respectively for 2001. Similarly, the linear regression relationships between percent stem end blue mold and P. expansum conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01 and 0.05, respectively). At the highest inoculum dose introduced into the settling tower, conidia per square centimeter and percent stem end blue mold at 8 months after inoculation were 39 and 26, respectively for 2000 and 66 and 23, respectively for 2003. Real-time PCR provided a rapid, quantitative measure of B. cinerea and P. expansum DNA on pear fruit surfaces. Because of possible year-to-year shifts in susceptibility of fruit to decay, disease incidence:inoculum dose relationships may be of most value compared within years rather than across years. This would facilitate comparison of decay risk among orchards in order to determine which fruit is most suitable for long-term storage.

Inoculum Sources of Botrytis cinerea Important to Pear Orchards in Oregon

Botrytis cinerea was capable of colonizing and persisting in tissue pieces of six common weed species found in pear orchards in Oregon from midsummer until pear harvest in early September. Sporulation occurred on blackberry mummies in orchard perimeters and ranged from 5,700 to 55,000 conidia per mummy at pear harvest. Conidia of B. cinerea also were observed on fresh blackberry fruit in 2 of 3 years. Sclerotia were present on only a few of the naturally infected pear fruit on the orchard floor through November; however, sclerotia production increased greatly between mid-November and mid-December. Over 90% of sclerotia from naturally infected, overwintered fruit germinated in the laboratory when collected 6 to 8 months after infection. Sclerotia formed on pear fruit inoculated with 28 different paired combinations of 22 isolates in the laboratory. Viability of sclerotia in inoculated pear fruit that overwintered on the orchard floor was 44 to 59% in March and April, and then declined steadily to about 25% at pear harvest. Germination of sclerotia was both sporogenic and myceliogenic. No apothecia were observed in the field or laboratory. An integrated approach to control is needed to deal with these multiple sources of potential inoculum.

Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of Rhodococcus fascians

Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.

First Report of Colletotrichum phormii Causing Anthracnose on New Zealand Flax in the United States

Phormium colensoi Hook.f. (syn. P. cookianum), New Zealand flax, (family Xanthorrhoeaceae) is popular in ornamental landscapes in the United States because of its sturdy blade-like foliage available in diverse colors. In February 2012, the Oregon State University Plant Clinic received three potted plants of P. colensoi Black Adder from a commercial nursery in Santa Cruz County, California. The margins and midribs of several leaves had brown lesions that were variable in size, and fusiform to ellipsoidal in shape. Embedded in the lesions were black acervuli without setae that exuded salmon-colored spore masses under moist conditions. Conidia were hyaline, cylindrical to fusiform, straight to slightly curved, and 22.4 to 35.2 × 4.0 to 6.4 (average 24.7 × 4.9) μm. Based on morphology, the fungus was confirmed by USDA-APHIS National Identification Services to be Colletotrichum phormii (Henn.) D.F. Farr & Rossman (2). In March 2012, the California Department of Food and Agriculture Plant Pest Diagnostic Lab received additional samples from the same nursery lot (25% disease incidence) from which a similar fungus was recovered. rDNA sequences of the internal transcribed spacer (ITS) region from the California isolate (GenBank KC122681), amplified using primers ITS1 and ITS4 (2), were 100% identical to multiple species of Colletotrichum, including C. phormii by a BLAST query (JQ948446 through JQ948453). ITS sequence similarity alone is not sufficient to address Colletotrichum taxonomy and must be used in combination with host range and morphology (1). Pathogenicity of C. phormii (isolate CDFA986) was tested on three Black Adder plants, which were inoculated with 6-mm agar plugs from a 14-day-old culture grown on half strength potato dextrose agar (PDA). Leaves were wound-inoculated along the midrib using colonized plugs (4). Five leaves per plant were inoculated with C. phormii plugs and five leaves per plant were treated with uncolonized PDA agar plugs as controls. Plants were sprayed with water and incubated in plastic bags at 22°C with a 12-h photoperiod. After 48 h, the bags and caps were removed and plants were kept under the same conditions. Two weeks later, water-soaked lesions had developed on the inoculated leaves. Lesions expanded along the midrib and became fusiform in shape after 21 to 28 days. C. phormii was isolated from lesion margins of all the inoculated leaves, but not from control leaves. This experiment was repeated once with similar results. Another Colletotrichum species, C. gloeosporiodes, also occurs on Phormium spp., but differs from C. phormii in morphology and symptom expression. Subsequent nursery and landscape surveys showed that anthracnose caused by C. phormii occurs on several P. colensoi cultivars as well as on P. tenax in five California counties including Santa Cruz, Yolo, Sacramento, San Luis Obispo, and Solano. C. phormii is also reported to infect P. colensoi and P. tenax in New Zealand, Europe, the United Kingdom, Australia, and South Africa (2,3). To our knowledge, this is the first report of C. phormii causing anthracnose on Phormium in North America. This disease could impact the American nursery trade and New Zealand flax production due to crop loss and increased production costs for pest management. References: (1) J. Crouch et al. Mycologia 101:648, 2009. (2) D. F. Farr et al. Mycol. Res. 110:1395, 2006. (3). H. Golzar and C. Wang. Australas. Plant Pathol. 5:110, 2010. (4) L. E. Yakabe et al. Plant Dis. 93:883, 2009.

First report of Phytophthora cactorum causing crown rot of Shepherdia x utahensis in the United States

Shepherdia argentea is a shrub native to the Intermountain West, often found in riparian areas. S. rotundifolia is also native, but limited in distribution to xeric environments in Arizona and southern Utah. The interspecific hybrid S. rotundifolia x argentea, named Shepherdia x utahensis, was made in an attempt to create an attractive, locally adapted, low water use landscape shrub. In fall of 2015, one of several failing young S. x utahensis plants from an ornamental native plant production and evaluation garden in Utah was submitted to the Oregon State University Plant Clinic for diagnosis. Additional plants from the site had already been destroyed. Symptoms included sparse foliage and extensive water-soaked necrosis in the root crown and lower branches. Tissue from the necrotic margins were surface disinfected (3 min, 10% household bleach (v/v)), rinsed, plated to 5% V8 modified PARP (0.1g/L dichloran substituted for pimaricin (PARD)) and incubated at 20°C. Canker margins were positive for Phytophthor...

First Report of Verticillium dahliae causing dieback of Highbush Blueberry (Vaccinium corymbosum) in Oregon and Washington.

In the spring of 2011, 2013, 2015, and 2016, cultivar Aurora, Blue Ribbon, Bluetta, and Cargo blueberry plants (Vaccinium corymbosum L.) from Oregon and Washington were sent to the Oregon State University Plant Clinic for diagnosis. Symptoms included foliar chlorosis, reddening, blackening of either part or the entire stem followed by stem dieback, vascular discoloration in the lower stems, and occasional wilt. The diseased plants were scattered in the fields, with incidence ranging from less than 5 to 40% of acreage. Isolations were made from surface-disinfected debarked stems and crown tissues to water agar and potato dextrose agar. Verticillium dahliae Kleb. was recovered from stems and crowns of all four cultivars, from which single-spore isolates were derived. Morphology of the conidiophores, conidia, and microsclerotia fit the description for V. dahliae (Klebahn 1913). Two of the isolates (OSU herbarium #OSC# 156080 and #OSC# 156081) were first identified by multiplex PCR assay (Inderbitzin et al. 2013) and subsequently sequenced. Partial DNA sequencing of the internal transcribed spacer (ITS) regions and TEF-1 alpha from both isolates were deposited in GenBank as accession numbers KY039311 and KY039312 (ITS) and KY039313 and KY039314 (TEF-1), and they showed 100% identity to the epitype strain of V. dahliae (ITS accession no. HQ206718, 461/461 and 466/466 bp for the two isolates, respectively; TEF-1 alpha accession no. HQ414624, 579/579 bp for both). Pathogenicity tests were performed by wounding and inoculating three lower stems each of three 2-year-old Bluetta blueberry plants with agar plugs of a 7-day-old single-spore culture of V. dahliae (#OSC# 156080). Three stems of another plant were wounded and inoculated with uncolonized agar plugs. Three months after inoculation, the stems had turned black up to 30 cm above inoculation sites, and the vascular tissue was discolored. Microscopic examination revealed masses of embedded V. dahliae microsclerotia along entire stems. Control plants remained symptomless, and V. dahliae was not recovered. Using the same two V. dahliae isolates as above, 15 cm tall blueberry plants of cultivars Bluetta, Earliblue, Draper, Polaris, and Rubel were inoculated by removing the bottom 1 cm of roots and dipping them in a conidial suspension (5 × 10⁶ conidia/ml) for 10 min, followed by planting in the greenhouse at 27°C. After 10 weeks, all inoculated cultivars showed defoliation, stunting, leaf reddening, vascular discoloration, and blackened stems. V. dahliae morphologically similar to our isolates was consistently recovered from stems of all inoculated cultivars, at least 2.5 cm above the soil line. Control plants remained healthy and did not yield V. dahliae. Verticillium-infected field-grown blueberries were largely from sites previously planted with peppermint or potato, crops susceptible to V. dahliae. Verticillium wilt has previously been reported on lowbush blueberry (V. angustifolium) in the United States and Canada (Brisson et al. 1976), on Vaccinium myrtillus (listed on PlantWise database), and on blueberry (Vaccinium sp.) in Chile (Cisternas and France 2009). None of these reports were confirmed by DNA evidence. To our knowledge, this is the first report of V. dahliae causing disease of highbush blueberry in the United States. Our results strongly suggest that fields with a history of V. dahliae infestation should not be planted with blueberry.