M. K. Hausbeck

Phytophthora capsici on Vegetable Crops: Research Progress and Management Challenges

Phytophthora capsici was first described by Leon H. Leonian at the New Mexico Agricultural Research station in Las Cruces in 1922 (65). In his report, he described a novel species of Phytophthora that caused considerable damage to chili pepper plants in the fall of 1918. A year later, the disease reappeared at the same site and also affected surrounding farms. During the late 1930s and early 1940s, recurrent problems with P. capsici in the Arkansas River Valley of Colorado were described on several vegetable hosts (51– 55,103). The first reported occurrence of P. capsici on a cucurbit crop occurred in 1937, when a 3.2-ha field of cucumbers became diseased resulting in 100% of the fruit rotting (51). By 1940, P. capsici had also been described on eggplant, honeydew melon fruit, summer squash, and tomato fruit (52,103). The disease on tomatoes was reportedly so severe that the viability of the processing tomato industry in the region was threatened. These early reports mirror the situation with P. capsici today on many modern vegetable production farms, especially those in the eastern United States (4,72,84,94). Our research was initiated in 1997, when crop losses caused by P. capsici threatened to bankrupt a number of vegetable producers in Michigan. Growers wanted to know why crop rotation and the use of fungicides in well-drained fields had not provided adequate protection against full-scale epidemics. At that time, there were fundamental gaps in our understanding of P. capsici’s epidemiology in Michigan, and it was difficult to answer these questions with any degree of certainty. We did not recognize the extent to which sexual recombination and genetic diversity could influence management options and success. In particular, the fungicide mefenoxam was being applied by some growers, and the sensitivity of natural populations of P. capsici in Michigan to mefenoxam was unknown at that time. Here we review recent advances in our understanding of P. capsici’s biology, in particular the role of sexual reproduction, and provide an overview of some of the management challenges presented by this information.

Mefenoxam Insensitivity and the Sexual Stage of Phytophthora capsici in Michigan Cucurbit Fields.

ABSTRACT The potential for outcrossing, occurrence of oospores, and inheritance of mefenoxam sensitivity was assessed in naturally occurring populations of Phytophthora capsici. Between 1997 and 1998, 14 farms were sampled, with 473 isolates recovered from cucurbit hosts and 30 from bell pepper. The A1 and A2 compatibility types were recovered in a roughly 1:1 ratio in 8 of 14 farms with sample sizes larger than 15. In 1997, one isolate was designated as insensitive and four as sensitive to mefenoxam. In 1998, 55% of the 498 isolates sampled were sensitive, 32% were intermediate, and 13% were fully insensitive to mefenoxam. In vitro characterization of mefenoxam sensitivity was conducted by crossing field isolates. Chi-square analysis of crosses between sensitive, intermediately sensitive, and insensitive isolates indicate that mefenoxam insensitivity segregated as an incompletely dominant trait unlinked to compatibility type (P = 0.05). Oospores were observed in diseased cucurbit fruit from four farms in 1998, and 223 oospore progeny were recovered from a single diseased cucumber. All six mefenoxam sensitivity-compatibility type combinations were present in these oospore progeny and within single fields. Based on these findings, we conclude that oospores likely play a role in the survival of P. capsici and that sexual recombination may significantly influence population structure.

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

Investigating the Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan

ABSTRACT Phytophthora capsici isolates were recovered from pepper and cucurbit hosts at seven locations in Michigan from 1998 to 2000. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity (MS), and amplified fragment length polymorphism (AFLP) marker profiles. In total, 94 AFLP bands were resolved. Individual populations were highly variable. Within populations, 39 to 49% of the AFLP bands were polymorphic and estimated heterozygosities ranged from 0.16 to 0.19. Of the 646 isolates fingerprinted, 70% (454) had unique AFLP profiles. No clones were recovered between years or locations. Pairwise F statistics (Phi(ST)) between populations from different locations ranged from 0.18 to 0.40. A tree based on unweighted pair-group method with arithmetic average cluster analysis indicates discrete clusters based on location. Isolates from the same location showed no clustering based on the year of sampling. Analysis of molecular variance partitioned variability among (40%) and within populations (60%). The overall estimated Phi(ST) was 0.34 (SD = 0.03). A1/A2 CT ratios were approximately 1:1, and MS frequencies were similar between years for the two locations sampled over time. These data suggest that P. capsici persists in discrete outcrossing populations and that gene flow among locations in Michigan is infrequent.

Fungal Planet description sheets: 107–127

Novel species of microfungi described in the present study include the following from Australia: Phytophthora amnicola from still water, Gnomoniopsis smithogilvyi from Castanea sp., Pseudoplagiostoma corymbiae from Corymbia sp., Diaporthe eucalyptorum from Eucalyptus sp., Sporisorium andrewmitchellii from Enneapogon aff. lindleyanus, Myrmecridium banksiae from Banksia, and Pilidiella wangiensis from Eucalyptus sp. Several species are also described from South Africa, namely: Gondwanamyces wingfieldii from Protea caffra, Montagnula aloes from Aloe sp., Diaporthe canthii from Canthium inerne, Phyllosticta ericarum from Erica gracilis, Coleophoma proteae from Protea caffra, Toxicocladosporium strelitziae from Strelitzia reginae, and Devriesia agapanthi from Agapanthus africanus. Other species include Phytophthora asparagi from Asparagus officinalis (USA), and Diaporthe passiflorae from Passiflora edulis (South America). Furthermore, novel genera of coelomycetes include Chrysocrypta corymbiae from Corymbia sp. (Australia), Trinosporium guianense, isolated as a contaminant (French Guiana), and Xenosonderhenia syzygii, from Syzygium cordatum (South Africa). Pseudopenidiella piceae from Picea abies (Czech Republic), and Phaeocercospora colophospermi from Colophospermum mopane (South Africa) represent novel genera of hyphomycetes. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Characterization of Phytophthora capsici from Michigan Surface Irrigation Water.

ABSTRACT Phytophthora capsici infects cucurbitaceous and solanaceous crops worldwide. In free water, P. capsici sporangia release zoospores that may be disseminated by moving surface water. Surface irrigation sources (river system, ponds, and ditches) in three Michigan counties with a history of P. capsici-susceptible crop production were monitored for the pathogen during four growing seasons (2002 to 2005). Pear and cucumber baits were suspended in water at monitoring sites for 3- to 7-day intervals and water temperature was recorded. Baits were washed and lesions were excised and cultured on water agar amended with rifampicin and ampicillin. P. capsici was detected at monitoring sites in multiple years, even when non-host crops were planted nearby. Recovered isolates (N = 270) were screened for sensitivity to the fungicide mefenoxam and characterized for mating type (MT). P. capsici isolates resistant to mefenoxam were common in water sources from southwest and southeast Michigan. Most monitoring sites yielded isolates of a 1:1 ratio of A1:A2 MTs. Amplified fragment length polymorphism analysis of select isolates from 2002 to 2004 indicated a lack of similarity groups persisting over time and in specific geographical locations. Data suggest that P. capsici did not overwinter in any of the surface water sources monitored. Water temperatures were correlated to positive P. capsici detection from all monitoring sites. The frequent detection of P. capsici in surface water used for irrigation in the primary vegetable growing regions in Michigan suggests that this is an important means of pathogen dissemination.

The Dynamics of Mefenoxam Insensitivity in a Recombining Population of Phytophthora capsici Characterized with Amplified Fragment Length Polymorphism Markers.

ABSTRACT Recent findings from Michigan suggest that recombination may play a role in the survival and evolution of sensitivity to the fungicide mefenoxam in populations of Phytophthora capsici on cucurbit hosts. In 1998, 63 mefenoxam insensitive isolates were recovered from a squash field in which mefenoxam had been applied. Additional isolates were recovered from untreated squash fields planted at this location in 1999 (200 isolates) and the spring of 2000 (34 isolates). Isolates from 1998 and 1999 were characterized using fluorescent amplified fragment length polymorphism (AFLP) markers and all isolates were screened for compatibility type and mefenoxam sensitivity. In 1998 and 1999, 92 and 71% of the isolates, respectively, had unique multilocus AFLP genotypes with no identical isolates recovered between years. Seventy-two identical AFLP markers were clearly resolved in both the 1998 and 1999 sample sets, and fixation indices for the 37 polymorphic AFLP loci indicate little differentiation between years. There was no decrease in the frequency of resistant isolates during the 2 years without mefenoxam selection. We conclude that oospores play a key role in overwintering and that the frequency of mefenoxam insensitivity may not decrease in an agriculturally significant time period (2 years) once mefenoxam selection pressure is removed.

The cucurbit downy mildew pathogen Pseudoperonospora cubensis.

UNLABELLED Pseudoperonospora cubensis[(Berkeley & M. A. Curtis) Rostovzev], the causal agent of cucurbit downy mildew, is responsible for devastating losses worldwide of cucumber, cantaloupe, pumpkin, watermelon and squash. Although downy mildew has been a major issue in Europe since the mid-1980s, in the USA, downy mildew on cucumber has been successfully controlled for many years through host resistance. However, since the 2004 growing season, host resistance has been effective no longer and, as a result, the control of downy mildew on cucurbits now requires an intensive fungicide programme. Chemical control is not always feasible because of the high costs associated with fungicides and their application. Moreover, the presence of pathogen populations resistant to commonly used fungicides limits the long-term viability of chemical control. This review summarizes the current knowledge of taxonomy, disease development, virulence, pathogenicity and control of Ps. cubensis. In addition, topics for future research that aim to develop both short- and long-term control measures of cucurbit downy mildew are discussed. TAXONOMY Kingdom Straminipila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Pseudoperonospora; Species Pseudoperonospora cubensis. DISEASE SYMPTOMS Angular chlorotic lesions bound by leaf veins on the foliage of cucumber. Symptoms vary on different cucurbit species and varieties, specifically in terms of lesion development, shape and size. Infection of cucurbits by Ps. cubensis impacts fruit yield and overall plant health. INFECTION PROCESS Sporulation on the underside of leaves results in the production of sporangia that are dispersed by wind. On arrival on a susceptible host, sporangia germinate in free water on the leaf surface, producing biflagellate zoospores that swim to and encyst on stomata, where they form germ tubes. An appressorium is produced and forms a penetration hypha, which enters the leaf tissue through the stomata. Hyphae grow through the mesophyll and establish haustoria, specialized structures for the transfer of nutrients and signals between host and pathogen. CONTROL Management of downy mildew in Europe requires the use of tolerant cucurbit cultivars in conjunction with fungicide applications. In the USA, an aggressive fungicide programme, with sprays every 5-7 days for cucumber and every 7-10 days for other cucurbits, has been necessary to control outbreaks and to prevent crop loss. USEFUL WEBSITES http://www.daylab.plp.msu.edu/pseudoperonospora-cubensis/ (Day Laboratory website with research advances in downy mildew); http://veggies.msu.edu/ (Hausbeck Laboratory website with downy mildew news for growers); http://cdm.ipmpipe.org/ (Cucurbit downy mildew forecasting homepage); http://ipm.msu.edu/downymildew.htm (Downy mildew information for Michigans vegetable growers).

Effect of Bactericides on Population Sizes and Spread of Clavibacter michiganensis subsp. michiganensis on Tomatoes in the Greenhouse and on Disease Development and Crop Yield in the Field

ABSTRACT Chemical applications, with the exception of mancozeb, reduced population sizes and spread of Clavibacter michiganensis subsp. michiganensis among tomato seedlings in the greenhouse and impacted subsequent plant development and yield in the field. While applications of copper hydroxide, copper hydroxide/mancozeb, copper hydroxide/mancozeb (premixed 12 h before spraying), streptomycin, and streptomycin/copper hydroxide to seedlings in the greenhouse did not differ significantly from the inoculated control, the trend was for these treatments to increase the survival of inoculated transplants in the field in comparison to the inoculated control. In the field, inoculated controls produced yields that were 63% (1995) and 51% (1996) of those produced by uninoculated controls. In both years, with the exception of mancozeb in 1995, all treatments resulted in yields similar to those obtained with the uninoculated control. Plant survival and yield in the field were severely affected when transplants had a pathogen population of >/= x 10(8) CFU/g of tissue. All treatments, with the exception of mancozeb, limited C. michiganensis subsp. michiganensis populations to <5.0 x 10(5). None of the treatments significantly reduced the incidence of fruit spotting compared with that of the inoculated control.

Effect of Crop Rotation on the Survival of Phytophthora capsici in Michigan

Phytophthora capsici is a limiting factor for some vegetable producers in Michigan despite crop rotation and fungicide applications. Our objective was to assess the effect of crop rotation on the survival of P. capsici at a naturally infested site in Michigan planted to cucumbers in 1998, corn in 1999 and 2000, and tomatoes in 2001. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity, and amplified fragment length polymorphism (AFLP) marker profiles, and compared with isolates recovered from other locations in Michigan during the same time period. The A1:A2 CT ratio was 1:1, and approximately 60% of the isolates recovered in both years were intermediately sensitive or insensitive to mefenoxam. The majority of the isolates (89%) had unique AFLP fingerprints, and no members of the same clonal lineage were recovered among years. Isolates from this location were more similar to each other than to isolates from other locations in Michigan, and it was not possible to distinguish isolates based on the year of isolation. Genetic similarity analyses indicate that isolates from this location are part of a genetically distinct outcrossing population. These data indicate that P. capsici persisted as oospores for the 2 years between cucumbers and tomatoes, and that crop rotation and mefenoxam are not likely to provide economic control.