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Verticillium dahliae causes Verticillium wilt, resulting in significant losses to potato production. Benzovindiflupyr, a succinate dehydrogenase inhibitor, effectively controls V. dahliae. However, frequent applications of the chemical may expedite the development of fungicide resistance in the pathogen population. To evaluate the risk of benzovindiflupyr resistance, 38 V. dahliae strains were obtained from diseased potatoes in Maine. The sensitivity of the field population was determined based on effective concentration for 50% inhibition (EC50), which ranged from 0.07 to 11.28 μg ml−1 with a median of 1.08. Segregated clusters of EC50 values indicated that Maine V. dahliae populations have developed benzovindiflupyr resistance. By exposing conidia of V. dahliae to a high concentration of benzovindiflupyr, 18 benzovindiflupyr-resistant mutants were obtained. To examine their fitness, the mutants were continuously subculture-transferred for up to 10 generations. Mycelial growth, conidial production, competitiveness, pathogenicity, and cross resistance of the 10th generation mutants were examined. Results showed that 50% of the resistant mutants retained an adaptive level in mycelial growth, and 60% maintained conidial production similar to their parents. Pathogenicity did not change for any of the mutants. No cross resistance was detected between benzovindiflupyr and either azoxystrobin, boscalid, fluopyram, or pyrimethanil. Thus, the resistance risk in V. dahliae to benzovindiflupyr should be considered in Maine potato production.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Sławiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch’s postulates were performed. Seed pieces of potato ‘Lamoka’ were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, ‘Lamoka’ tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the United States since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the United States outbreak were assembled and compared with 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. The pangenome of the 32 strains contained 6,693 genes, 3,377 of which were core genes. By screening primary protein subunits associated with virulence from all U.S. strains, we found that many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes, were highly conserved but that type IV and type VI secretion system genes varied. The clade I strains encoded two clusters of type IV secretion systems, whereas the clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than did clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant in the U.S. outbreak.

An outbreak of blackleg and soft rot of potato, caused primarily by the bacterial pathogen Dickeya dianthicola, has resulted in significant economic losses in the northeastern United States since 2015. The spread of this seedborne disease is highly associated with seed distribution; therefore, the pathogen likely spread with seed tubers. To describe the blackleg epidemic and track inoculum origins, a total of 1,183 potato samples were collected from 11 states associated with blackleg outbreak from 2015 to 2019. Of these samples, 39.8% tested positive for D. dianthicola. Seventeen isolates of D. dianthicola were recovered from these samples and the genetic diversity of these isolates was examined. Fingerprinting with BOX-A1R-based repetitive extragenic palindromic PCR and phylogenetic analysis based on sequences of the 16S rRNA and gapA genes indicated that D. dianthicola isolates were divided into three genotypes, denoted types I, II, and III. Ninety-five percent of samples from Maine were type I. Type II was found in Maine only in 2015 and 2018. Type II was present throughout the 5 years in some states at a lower percentage than type I. Type III was found in Pennsylvania, New Jersey, and Massachusetts, but not in Maine. Therefore, type I appears to be associated with Maine, but type II appeared to be distributed throughout the northeastern United States. The type II and rarer type III strains were closer to the D. dianthicola type strain isolated from the United Kingdom. This work provides evidence that the outbreak of blackleg of potato in the northeastern United States was caused by multiple strains of D. dianthicola. The geographic origins of these strains remain unknown.

Dickeya dianthicola (Samson) is an important pathogen causing blackleg disease of potato. Previous work suggested that insects might vector species of Pectobacteriaceae between plants but no conclusive work has confirmed this. Green peach aphids (Myzus persicae Sulzer) and Colorado potato beetles (Leptinotarsa decemlineata Say) are aggressive potato pests and related to known vectors of several species of bacteria other than D. dianthicola. This study sought to determine whether these insects vector D. dianthicola for potato infection. Neither insect species showed olfactory discrimination based on the presence of infection in laboratory tests but beetles were repelled by uninfected foliage treated with 2,3-butanediol, a primary Dickeya metabolite. Beetle recruitment to plants was not affected by their infection status; however, aphids preferred uninfected foliage when conspecifics were present. In the laboratory, neither insect acquired or transmitted D. dianthicola through feeding. In the field, neither insect’s abundance was significantly correlated with disease spread. Overall, this study did not find indications that D. dianthicola is vectored by the tested insect species. Therefore, efforts to limit Dickeya spread should focus on sanitation, water management, and seed screening—not on the control of these insect species. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Rhizoctonia solani is a widely distributed soilborne plant pathogen, and can cause significant economic losses to crop production. In chemical controls, SYP-14288 is highly effective against plant pathogens, including R. solani. To examine the sensitivity to SYP-14288, 112 R. solani isolates were collected from infected rice plants. An established baseline sensitivity showed that values of effective concentration for 50% growth inhibition (EC50) ranged from 0.0003 to 0.0138 μg/ml, with an average of 0.0055 ± 0.0030 μg/ml. The frequency distribution of the EC50 was unimodal and the range of variation factor (the ratio of maximal over minimal EC50) was 46.03, indicating that all wild-type strains were sensitive to SYP-14288. To examine the risk of fungicide resistance, 20 SYP-14288-resistant mutants were generated on agar plates amended with SYP-14288. Eighteen mutants remained resistant after 10 transfers, and their fitness was significantly different from the parental strain. All of the mutants grew more slowly but showed high virulence to rice plants, though lower than the parental strain. A cross-resistance assay demonstrated that there was a positive correlation between SYP-14288 and fungicides having or not having the same mode of action with SYP-14288, including fluazinam, fentin chloride, fludioxonil, difenoconazole, cyazofamid, chlorothalonil, and 2,4-dinitrophen. This result showed a multidrug resistance induced by SYP-14288, which could be a concern in increasing the spectrum of resistance in R. solani to commonly used fungicides.

Potato common scab is an important soilborne disease worldwide that can significantly reduce the quality and economic values of potato. The disease is caused by multiple species of Streptomyces, which are not well controlled due to lack of effective strategies. Streptomyces galilaeus has been recently identified as a dominant species causing potato common scab in Inner Mongolia, China. This study was focused on screening and characterizing antagonists for biological control against pathogenic S. galilaeus. Bacterial strain PBSH9 was isolated from a potato tuber. PBSH9 was identified as a Streptomyces sp. on the basis of morphological, physiological, and biochemical characteristics, as well as DNA sequence analysis. PBSH9 inhibited S. galilaeus with a diameter of inhibitory zone of 19.8 mm on agar plates. The extracellular filtrate of PBSH9 also inhibited S. galilaeus growth with a diameter of inhibition zone of 10.0 mm. Furthermore, PBSH9 promoted potato sprouting and emergence. Disease control was up to 81.88% in greenhouse trials, and from 47.64 to 73.97% in 3-year field trials. Among the tested inoculation methods, seed treatment was more effective than soil drenching for PBSH9 application. PBSH9 not only effectively controlled potato common scab but also increased potato growth. Thus, it can be a potential candidate for biocontrol agent.

Spiral plating technique is reliable, repeatable, and more efficient than dilution plating methods in studying the efficacy of antimicrobial products. In this method, the concentration of chemicals can be varied at different positions on agar plates, but its calculation requires using a commercial software. To establish a user-friendly and cost-free platform, the R package ECX was developed to calculate chemical concentrations in spiral plating technique. Mathematical models were established for calculating dispensed volume on agar plates using variables (molecular weight and agar height) that affect diffusion. In addition to the R packages, the web-based Shiny extensions ECX, multi, and ppm were developed to provide a graphical interface for calculating individual concentrations, multiple concentrations, and stock concentrations, respectively. No significant differences were observed (P > 0.05) when ECX was compared with the commercial software. The ability to import and process large datasets makes the ECX package a better option for spiral plating technique studies. Furthermore, the multiplatform nature of the ECX package overcomes limitations presented in other software. Therefore, these ECX characteristics can increase the use of the spiral plating technique for sensitivity studies. Accepted for publication 21 June 2016.

Pink fruit rot of peach (Prunus davidiana) was observed in an orchard near Huairou, Beijing, China, in 2018. Approximately 90% of peach trees were infected, and diseased fruits were covered with pink-colored mold. To identify the causal agent, diseased fruits were collected, and fungal isolates were obtained from symptomatic fruit tissue. Initially, on potato dextrose agar, the isolates produced thin mycelium, with rare aerial hyphae at 28°C. After 3 days of incubation, the mycelium became pink and produced numerous powdery conidia. Conidia were pyriform, ovate, with papillary protuberances at one end. They were colorless individually but pinkish in mass, with one septation that slightly constricted the middle of the spore. The conidia had an average size of 15 × 6 μm under a light microscope. Two isolates, HB1-1a and HB1-2c, were used in the pathogenicity tests. Each isolate was inoculated onto 10 healthy P. davidiana fruit by pipetting with 100 μl of spore suspension at the concentration of 1 × 10⁶ conidia/ml. Distilled water served as the control. All treated fruits were enclosed in a plastic bag and stored at 28°C under a 12-h light/dark cycle. After 15 days of incubation, symptoms similar to those observed in the orchard developed. The same fungus was reisolated from the diseased fruit. To further confirm the identity of the pathogen, the internal transcribed spacer (ITS) and part of the 5′ end of the β-tubulin (TUB) genes of the isolates HB1-1a and HB1-2c were amplified by using two different primer sets: ITS1/ITS4 (White et al. 1990) and T1/T22 (Hamid et al. 2014). The PCR products had sizes of approximately 700 bp for ITS and 1.5 kb for TUB. The ITS sequence of isolates HB1-1a and HB1-2c (GenBank nos. MN629234 and MN629235) indicated 99.67% identity with Trichothecium roseum (GenBank no. EU552162.1). The partial β-tubulin gene sequence (GenBank nos. MN640076 and MN640077) showed a 100% homology with T. roseum strain UoS09 (GenBank no. KJ607590.1). Therefore, this fungus was identified as T. roseum based on morphological characteristics and molecular data. T. roseum is reported to have a wide range of host (Hong et al. 1997; Inacio et al. 2011). In China, this fungus was first to be found in apple fruit introduced by a traveler in 2018 (Xu et al. 2018). To our knowledge, this is the first report of T. roseum causing pink fruit rot of P. davidiana in China.

A survey of seed potato tubers in Michigan seed production storage facilities was carried out during 2009 and 2010. Fusarium spp. associated with tuber dry rot symptoms were identified to species and tested for sensitivity to difenoconazole, fludioxonil, and thiabendazole. Symptomatic tubers (n = 370) were collected from a total of 51 seed lots, from which 228 isolates of Fusarium were recovered and identified to 11 species. Fusarium oxysporum was the most commonly isolated species (30.3%), followed by F. equiseti (19.3%). F. sambucinum and F. avenaceum were third most prevalent (each at 13.6%). Less prevalent species (each at 4 to 10%) included F. cerealis, F. solani, and F. acuminatum; and species present at ≤3% included F. sporotrichioides, F. torulosum, F. tricinctum, and F. graminearum. Representative isolates of all species were pathogenic when inoculated onto seed tubers (‘Dark Red Norland’). Isolates of F. sambucinum were the most virulent. All 228 isolates of Fusarium were sensitive to difenoconazole (effective fungicide concentration that caused 50% inhibition of mycelial growth [EC50] < 5 mg/liter). Insensitivity to fludioxonil (EC50 > 100 mg/liter) was detected only for F. sambucinum and F. oxysporum isolates at 8.9 and 20.4%, respectively. All isolates were sensitive to thiabendazole (EC50 < 5 mg/liter), except for those of F. sambucinum (EC50 > 100 mg/liter). Therefore, knowledge of what Fusarium spp. are present in seed potato storage facilities in Michigan may be important if using fludioxonil or thiabendazole for seed piece treatment but not when using difenoconazole.

Bi, Y., Jiang, H., Hausbeck, M. K., and Hao, J. J. 2012. Inhibitory effects of essential oils for controlling Phytophthora capsici. Plant Dis. 96:797803. Essential oils (EOs) were studied in vitro and in vivo for inhibiting Phytophthora capsici. Mycelial growth of P. capsici was examined on EO-amended media or after exposing it to EO volatiles. The efficacy of EOs was determined by estimating the effective concentration for 50% inhibition of P. capsici mycelial growth (EC50). Among 14 tested commercial products, oregano, palmarosa, and red thyme EOs had the lowest EC50 values (

Potato common scab, caused by Streptomyces spp., is an annual production problem for potato growers, and not effectively controlled by current methods. A field with naturally occurring common scab suppression has been identified in Michigan, and confirmed to have a biological basis for this disease suppression. This field and an adjacent scab nursery conducive to disease were studied using pyrosequencing to compare the two microbial communities. Total DNA was extracted from both the disease-conducive and -suppressive soils. A phylogenetically taxon-informative region of the 16S rRNA gene was used to establish operational taxonomic units (OTUs) to characterize bacterial community richness and diversity. In total, 1,124 OTUs were detected and 565 OTUs (10% dissimilarity) were identified in disease-conducive soil and 859 in disease-suppressive soil, including 300 shared both between sites. Common phyla based on relative sequence abundance were Acidobacteria, Proteobacteria, and Firmicutes. Sequences of Lysobacter were found in significantly higher numbers in the disease-suppressive soil, as were sequences of group 4 and group 6 Acidobacteria. The relative abundance of sequences identified as the genus Bacillus was significantly higher by an order of magnitude in the disease-conducive soil.

Bacterial soft rot and blackleg of potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. can be a problem that causes severe economic losses (Jiang et al. 2016). A survey was conducted in 10 Maine potato fields from May to August in 2015, following the outbreak of blackleg in the Northeastern US (Jiang et al. 2016). Potato plants with blackleg symptoms and darkened and necrotic basal stems were collected from Maine potato fields in 2015. The lower 2 cm of blackleg symptomatic stems were excised, surface sanitized with 75% ethanol for 40 seconds, transferred to a 0.65% sodium hypochlorite solution for 1 min, followed by rinsing with sterile distilled water. The stem segment was then placed in a 50-mL tube containing 10 ml of sterile distilled water for 5 min to allow the bacteria to release from the plant tissue. The bacterial suspension was plated on crystal violet polypectate agar (Helias et al. 2012), and single colonies that formed cavities after 3 days were selected, purified, and ...

A novel strain of Streptomyces (named DS3024) was isolated from a potato field in Michigan in 2006. The taxonomy of the organism was determined by morphology, biochemistry, and genetic analysis. Analysis of the 16S ribosomal RNA gene sequence indicated that the organism was most similar to an isolate of Streptomyces sp., ME02-6979.3a, which is not pathogenic to potato tubers but is distinct from other known pathogenic Streptomyces spp. Strain DS3024 has genes that encode thaxtomin synthetase (txtAB), which is required for pathogenicity and virulence, and tomatinase (tomA), which is a common marker for many pathogenic Streptomyces spp. However, the nec1 gene (associated with virulence in most pathogenic Streptomyces spp.) was not detected. The new strain was capable of growth at pH 4.5, caused necrosis on potato tuber slices, and produced thaxtomin A. In greenhouse experiments, DS3024 caused scab symptoms on potato tubers similar to those caused by Streptomyces scabies on tubers of potato cv. Atlantic, which is scab susceptible. We propose that DS3024 is a new strain of Streptomyces capable of causing common scab on potato tubers. The prevalence of this strain of Streptomyces in potato-producing areas in the north-central United States has not been determined.

Hao, J. J., Yang, M. E., and Davis, R. M. 2009. Effect of soil inoculum density of Fusarium oxysporum f. sp. vasinfectum race 4 on disease development in cotton. Plant Dis. 93:1324-1328. The effect of soil inoculum density (0 to 106 conidia/g of potting mix) of Fusarium oxysporum f. sp. vasinfectum race 4 on plant growth (weight, height, and number of nodes), Fusarium wilt symptoms, vascular discoloration, and number of CFU per gram of stem tissue was determined on five cotton cultivars grown in a greenhouse. In the susceptible cv. DP744, symptoms of wilt and reductions in plant growth occurred at inoculum levels of 103 conidia/g of potting mix and higher, whereas plant growth of the resistant Pima cv. Ph800 was not affected by any soil inoculum densities. Cvs. DP340, Ph72, and UltEF all responded similarly, with a relatively moderate negative growth response to soil inoculum densities generally beginning at 10 4 conidia/g of potting mix. F. oxysporum f. sp. vasinfectum race 4 was recovered from the stems of all cultivars by plating macerated stem tissue on a semiselective medium. There was a significant positive correlation between CFU and inoculum density above 104 conidia/g of potting mix for DP744 and UltEF. The fungus was recovered relatively infrequently from stems of Ph 800 plants inoculated with any inoculum density of F. oxysporum f. sp. vasinfectum. Fusarium wilt caused by F. oxysporum f. sp. vasinfectum race 4 is an inoculum density- dependent disease; the disease was generally mild and the fungus was rarely recovered from stems at inoculum levels less than 104 conidia/g of potting mix.

The ability of soil-applied garlic powder and diallyl disulfide to stimulate germination of sclerotia of Sclerotium cepivorum, the cause of white rot of onion and garlic, was evaluated in four field trials. Because sclerotia germinate in response to exudation of specific volatile sulfides and thiols from allium roots, sulfides applied to the ground in the absence of an allium crop cause death of the sclerotia after they germinate and exhaust nutrient reserves. In this study, garlic powder and a synthetic garlic oil, diallyl disulfide, were incorporated into the soil in commercial fields naturally infested with S. cepivorum. Methyl bromide was included as a chemical control. Within 3 months after treatment, over 90% of the sclerotia died in the plots treated with the germinationstimulants, which was similar to the reduction of viable sclerotia achieved with an application of methyl bromide. The degree of sclerotial mortality in plots treated with garlic powder at 112 kg/ha or more was almost equal to that achieved by diallyl disulfide at 0.5 ml/m2 or methyl bromide at 448 kg/ha. Despite the efficacy of the stimulants and methyl bromide to reduce populations of sclerotia, the pathogen caused substantial root rot and yield losses in subsequent garlic crops planted about a year after soil treatment. However, germination stimulants have utility because the reduction of the vast majority of sclerotia in a field reduces the risk of spread of the pathogen to neighboring fields.

Field experiments were conducted at Spence Road site and at the Hartnell College East Campus site in Salinas, CA, to determine the effects of crop rotation with broccoli or a fallow period on lettuce drop caused by Sclerotinia minor and the density of pathogen sclerotia in the soil. Treatments at the Spence Road site with low inoculum density (3 of soil) distributed randomly included: successive crops of lettuce (LLL), lettuce rotated with broccoli (LBL), and lettuce followed by a fallow period (LFL). Treatments at the Hartnell site with high inoculum density (>7 sclerotia per 100 cm3 of soil) distributed uniformly included: continuous lettuce (LLLL), broccoli-lettuce-broccoli-lettuce (BLBL), broccoli-broccoli-lettuce-lettuce (BBLL), and fallow-lettuce-fallow-lettuce (FLFL). At the Spence Road site, continuous lettuce did not increase lettuce drop incidence for at least 2 years, although an increase in soilborne sclerotia was observed annually but was below the threshold at which a correlation between inoculum density and disease incidence is observed. Rotation with broccoli resulted in small reductions in disease incidence only in the first year. The density of sclerotia was lowest in the LFL treatment, and the highest in the LLL. At the Hartnell site, rotation with broccoli significantly reduced both sclerotia and lettuce drop incidence. The number of broccoli crops rather than the sequence of lettuce rotations with broccoli was critical for reducing the numbers of S. minor sclerotia in soil. Fallowing after a lettuce crop resulted in marginal reductions in sclerotia and lettuce drop incidence. Viability of recovered sclerotia was not significantly different between treatments, although differences between seasons were detected. Results suggest that rotations with broccoli can be a practical lettuce drop management strategy.

The spatial patterns of microsclerotia of Verticillium dahliae in soil and wilt symptoms on cauliflower were determined at three sites in each of two fields in 1994 and 1995. Each site was an 8 × 8 grid divided into 64 contiguous quadrats (2 by 2 m each). Soil samples were collected to a depth of 15 cm with a probe (2.5 cm in diameter), and samples from four sites in each quadrat were bulked. Plants in each quadrat were cut transversely, and the number of plants with vascular discoloration and the number without discoloration were recorded. The soil was assayed for microsclerotia by the modified Anderson sampler technique. Lloyd's index of patchiness (LIP) was used as an indicator to evaluate the aggregation of microsclerotia in the field. Spatial autocorrelation and geostatistical analyses were also used to assess the autocorrelation of microsclerotia among quadrats. The LIP for microsclerotia was greater than 1, indicating aggregation of propagules; however, the degree of aggregation at most sites was not high. Significant autocorrelation within or across rows was detected in some spatial autocorrelograms of propagules, and anisotropic patterns were also detected in some oriented semivariograms from geostatistical analyses for microsclerotia, indicating the influence of bed preparation in the fields on pathogen distribution. The parameter estimates p and θ in the beta-binomial distribution and the index of dispersion (D) associated with the distribution were used to assess the aggregation of diseased plants at each site. A random pattern of wilt incidence was detected at 7 of 12 sites, and an aggregated pattern was detected at 5 of 12 sites. The degree of aggregation was not high. A regular pattern of wilt severity was detected at all sites. The high disease incidence (77 to 98%) observed at 11 of the 12 sites could be explained by high inoculum density.

During a survey of carrot (Daucus carota L.) cavity spot in Michigan in September 2010, carrot roots with typical cavity spot symptoms were collected from production fields in Fremont Co. The lesions were excised from infected roots, surface-disinfested with 0.62% NaClO for 3 min, rinsed in sterilized, distilled water three times, cut into 0.5 cm long pieces, and then plated on water agar (WA) amended with carbendazim (10 μg/ml), ampicillin (50 μg/ml), rifampicin (50 μg/ml), and pentachloronitrobenzene (10 μg/ml) (cumulatively referred to as CARP). Plates were incubated at 22 ± 1°C in the dark for 3 days. Pure cultures of the isolates were obtained by transferring a single hyphal tip of each colony to potato dextrose agar (PDA) amended with CARP. Among the 33 isolates obtained, M2-05 was identified as a Pythium sp. that differed from the known cavity spot pathogens of carrot. The isolate has spherical hyphal swellings but no other distinguishing morphological characteristics. M2-05 was further classified by analyzing the partial sequences of four genes: the internal transcribed spacer (ITS) region of ribosomal DNA, beta-tubulin (β-tub), cytochrome c oxidase subunit 2 (cox 2), and NADH dehydrogenase subunit 1 (nadh 1) (1,3). A BLAST search of these sequences for M2-05 was conducted using the nucleotide database of GenBank, resulting in 100% similarity to all four sequenced genes of P. recalcitrans (2). The DNA sequences of M2-05 were deposited in GenBank (JQ734349, JQ734229, JQ734289, and JQ734409 for ITS, β-tub, cox 2, and nadh 1, respectively). Koch's postulates were conducted by inoculating mature carrot roots (cv. Nantindo) with mycelial plugs (4 mm in diameter) cut from the margin of actively growing colonies of M2-05 on PDA plates. Two mycelial plugs were placed on each carrot root at 3-cm intervals, with the mycelial side facing the root; and two non-colonized agar plugs were placed similarly for the non-inoculated control treatment. In comparison, carrot roots also were inoculated with an isolate of each of P. sulcatum and P. violae using the same method. There were four replicate carrot roots inoculated for each isolate and each of the control treatments. The inoculated roots were placed on a plastic grid (7 mm in height) in a closed plastic container, with moist paper towels underneath the grids. The container was incubated in the dark at 22 ± 1°C, and the roots were sprayed gently daily with sterilized, distilled water to maintain high humidity. Brown lesions were observed on all inoculated carrot roots 5 days after inoculation. The lesions measured 0.68 ± 0.48, 1.20 ± 0.71, and 0.56 ± 0.31 mm2 averaged over all eight lesions for the isolates of P. recalcitrans, P. sulcatum, and P. violae, respectively. Symptomatic tissues from the inoculated roots were excised and incubated on WA-CARP plates, and the culture from each lesion confirmed as the isolates inoculated using the same molecular methods described above. The carrot tissue under the control agar plugs remained symptomless, and no Pythium was recovered from the control roots. P. recalcitrans was described in 2008 as infecting roots of Beta vulgaris and Vitis vinifera (2). To our knowledge, this is the first published report of P. recalcitrans naturally infecting carrot roots, not only in Michigan, but anywhere in the world. References: (1) L. Kroon et al. Fungal. Genet. Biol. 41:766, 2004. (2) E. Moralejo et al. Mycologia 100:310, 2008. (3) N. O. Villa et al. Mycologia 98:410, 2006.