Characterization of Phytophthora capsici from Michigan Surface ...

nearby field were sampled (foliage, stems, tubers, and associatedsoil), but the pathogen was not detected. At SW2 to SW6 sites,when a nearby field was planted to cucumber, P. capsici diseasesymptoms were observed and included fruit with water-soakedlesions and pathogen sporulation (Table 1).Ponds. P. capsici was detected in a naturally fed pond (NW1)and a pond fed by a deep well (NW3). In a second well-fed pond(NW2) with nearby field sites historically infested with P. capsici,the pathogen was not detected. Baiting in the naturally fed pond(NW1) was initiated late in the 2002 growing season in responseto crop loss in an adjacent field due to P. capsici. The pathogenwas detected immediately and throughout the remainder of thegrowing season. P. capsici was isolated from a nearby field ofinfected yellow squash in 2002 and from snap beans in 2003(Table 1). No P. capsici was recovered from sampled carrots in2004 (Table 1). Well-fed pond, NW3, had just a single baitingperiod with positive P. capsici detection.Ditches. In 2004, monitoring was initiated in response to widespreadand severe disease in a hand-harvested cucumber field inSE Michigan (Lenawee County). The detection of P. capsici atthis site (SE1: run-off ditch) represents the first confirmation ofthis pathogen in water used for irrigation in SE Michigan. Despiteour interest in continuing to monitor this site in 2005, the growerdeclined. The 2005 NW Michigan ditch site (NW4: culvert ditch)represented a culvert that received run-off from a number offields, some with a known history of P. capsici infestation. Thepathogen was detected immediately following baiting initiationand throughout the remainder of the growing season (Table 1).MTs. For all monitoring sites where more than one P. capsiciisolate was obtained, both A1 and A2 MTs were typically detected(Table 2). In several sites, the ratio of A1 to A2 was nearly 1:1when totaled over the monitored years. At sites where the ratiowas not close to a 1:1, the A1 MT occurred more frequently whenexamined across the isolate totals for a particular site (Table 2).Mefenoxam sensitivity. Each year, river sites yielded P. capsiciisolates of each of the mefenoxam sensitivity categories (sensitive[S], intermediately sensitive [I], and resistant [R]) (Table 2).Approximately half of the P. capsici isolates from the river sitesin 2002 were I with one quarter of the isolates R (Table 2). Mostisolates from 2003 and 2004 were R (Table 2). In the last year ofmonitoring (2005), most (88%) isolates were S (Table 2).Nearly all P. capsici isolates collected from the NW Michiganregion (Oceana County) during 2002 to 2005 were S (Table 2). In2002, there was a single I isolate collected (Table 2). The culvertditch monitored in 2005 yielded 14 P. capsici isolates, 13 of whichwere S to mefenoxam (Table 2). The majority of the P. capsiciisolates from the run-off ditch monitored in 2004 in SE Michigan(Lenawee County) collected were I and approximately 30% wereR (Table 2).Water temperature. Water temperatures were significantlycorrelated to positive P. capsici detection from the monitoringsites from 2002 to 2005 when analyzed using a nonlinear quadraticpolynomial regression (R 2 = 0.9408) (Fig. 4). Most (92%) ofthe baiting periods yielding P. capsici occurred during times whenwater temperatures were between 15 and 25°C (Table 3; Fig. 4).P. capsici was not identified from water sources when temperaturesfell below 14°C or rose above 25°C (Table 3). Average watertemperatures at the river monitoring sites during baiting periodswith positive P. capsici detection were 17 to 19°C. At the pondsites, average water temperatures during baiting periods withP. capsici infestation were 20 to 22°C. The run-off and culvertditch sites had average water temperatures of 21°C during baitingperiods with positive P. capsici detection.Fig. 2. Amplified fragment length polymorphism similarity tree of Phytophthoracapsici isolated from Michigan surface water. Long-term numbers (LT#)refer to culture numbers maintained in the laboratory of M. K. Hausbeck atMichigan State University. Mating types (MT) are either A1 or A2.Mefenoxam sensitivity (MS) characteristics are sensitive (S), intermediatelysensitive (IS), and resistant (R).Fig. 3. Schematic of six sites along a river and creek system of southwestMichigan that were monitored for Phytophthora capsici infestation during thegrowing seasons of 2002 through 2005. Site 1 is a creek that flows into theriver. Arrows indicate direction of water flow.424 PHYTOPATHOLOGY

Rainfall. For the river system, total rainfall accumulation wasgreatest in 2003 and 2004 (Fig. 5). During baiting periods whenP. capsici was detected at river sites, rainfall accumulationaveraged

organisms were also isolated from both pear and cucumber fruitsduring the 4 years of monitoring. Based on morphologicalcharacteristics, Pythium spp. were identified, to genus only, eachyear and were prevalent during the early (April and May) and late(September and October) portions of the growing season (data notshown). P. citricola was identified to species by sequence analysisof the internal transcribed spacer 1 (ITS1) and ITS2 spacerregions (data not shown). Other Phytophthora spp. were identifiedto genus based on morphological characteristics.Water temperature exhibited some influence on the frequencyof P. capsici detection. In our study, regression analysis determinedthat water temperatures were correlated with P. capsiciincidence on pear and cucumber baits (R 2 = 0.9408). Thereappeared to be a lower temperature threshold (14°C) at whichP. capsici was not detected. When lupines were used to bait forPhytophthora spp. in cranberry irrigation reservoirs in NewJersey, a significant positive correlation was observed betweenwater temperature and detection of P. cinnamomi; a negative relationshipwas observed for temperature and P. megasperma (34).TABLE 3. Average water temperature (°C) during baiting periods from allsites and years (2002 to 2005) and the number of baiting periods with positivedetection of Phytophthora capsiciTemperatureduring baiting (°C)Total no. ofbaiting periodsNo. of baiting periodswith P. capsiciIncidence(%)5–10 24 0 010–15 114 6 815–20 277 47 1720–25 120 21 1825–30 8 0 0Fig. 5. Rainfall (weekly totals) and Phytophthora capsici detection data forSW river monitoring sites in Allegan County, Michigan, in A, 2002, B, 2003,C, 2004, and D, 2005. Gray vertical bars indicate rainfall (mm). Stars indicatepositive P. capsici detection.P. palmivora and P. citrophthora have been shown to remain motilein water at an optimal temperature of 17 and 12.5°C, respecttively(7). The length of time zoospores can continue to swim andremain viable may be impacted by water temperature (7,8).Further laboratory studies may elucidate the relationship betweenwater temperature and P. capsici zoospore activity. In Michigan,low water temperature typically occurs at a time when crops maynot be established (early spring) or have been removed (earlyfall).Frequency of P. capsici detection was greatest at river and ditchsites and rare at pond sites fed by deep wells. Although riverwater can have many potential points of infestation and is anunreliable source for clean irrigation water, ponds may alsoharbor P. capsici. The naturally fed pond, NW1, yielded P. capsiciin each of the 4 years monitored despite the planting of non-hostcrops in the nearby field in two of these years. One of the twowell-fed ponds (NW3) yielded just one P. capsici isolate duringour monitoring. Ponds fed by deep wells are a safer option forgrowers than using surface water for irrigation, provided that runofffrom nearby fields does not enter the holding pond. Theditches in our study received direct run-off of water fromnumerous infested fields and yielded P. capsici for nearly everyperiod monitored. Both ditch sites fed into water retention/pumpingponds used for irrigation.Rain and/or irrigation events likely prompt run-off from fieldswith P. capsici-infected crops into ditches, rivers, and creeks.However, precipitation data do not clearly indicate pathogendetection following significant rainfall. Grower irrigation datawere not collected and may have impacted surface water infestation.Michigan cucurbit growers attempt to provide an average of1 in. of water to their crops per week during the summer growingmonths. Monitoring water during the host crop-growing seasonindicated that irrigation water was often infested when the need toirrigate crops was highest, typically in late July and August,increasing the risk for pathogen dissemination.P. capsici was most frequently detected in water at monitoringsites during years when crown and fruit rot was observed in anearby field. However, P. capsici was consistently detected in irrigationsources even after 1 to 3 years without a host crop growingnearby. P. capsici can persist within the environment outside ofhost tissue (9,23,37). In Michigan, molecular techniques haveconfirmed that P. capsici oospores can survive dormantly in soilfor 3 years (23). Weed species have been identified as alternativehosts for P. capsici under Florida field conditions (9,35). P. capsicimay be a weak pathogen of weed species or may simplycolonize weed root systems (9). In addition, rotational crops maynot be serving to limit the overall soil population of the pathogen.Snap beans were increasingly grown in rotation to vegetable cropsin Michigan, prior to 2003. Unfortunately, snap beans have sincebeen determined to be susceptible to P. capsici under fieldconditions in Michigan and other vegetable-producing regions ofthe United States (11).The consistent presence of the pathogen in moving surfacewater without a nearby infected host crop may indicate a longrange of movement of P. capsici. In a study of the movement of aPhytophthora sp. (interspecific hybrid of P. cambivora and anunknown Phytophthora sp. related to P. fragariae) on alder inBavaria, it was determined that once the pathogen was introducedinto a river system upstream, it infected alders downstream (17).In addition, area nurseries that had high incidence of infectedalder seedlings were found to be using water from infested rivercourses for irrigation (17). Due to the proximity of the Michiganriver system to susceptible crop production, there are numerouspotential sources of P. capsici infestation. Drainage tiling mayalso be responsible for the movement of infested water. Subsurfacedrainage tiling, for removal of excess field water, occursthrough a network of perforated plastic tubes installed below thesoil surface. Water from the tiles eventually drains into ditches,426 PHYTOPATHOLOGY

etention ponds, rivers, creeks, and lakes. It is difficult to estimatehow far P. capsici can move via water based on the data presentedhere because we did not detect identical AFLP genotypes frommultiple locations. The identical genotypes (similarity subgroups)recovered from individual sites likely represented the dispersal ofasexual sporangia and zoospores, as P. capsici sporulates heavilyon host crops.Isolates of P. capsici resistant to mefenoxam may be disseminatedby irrigation. The extensive use of phenylamidefungicides for disease management can render P. capsici fieldpopulations resistant (19–21). Growers in SW Michigan have along history of phenylamide fungicide use (mefenoxam and/ormetalaxyl) compared with growers in NW Michigan. Mefenoxamhas been relied upon only in recent years to manage vegetablediseases in the SE region. Characterization of populations ofP. capsici collected from SW Michigan from 1997 to 2001 indicateda high incidence of mefenoxam insensitivity (21), whereasisolates from NW Michigan were sensitive to the fungicide (20).In the first monitoring year (2002), most isolates obtained fromthe river sites in SW Michigan were intermediately sensitive tothe fungicide mefenoxam; in the subsequent 2 years, approximatelyhalf of the isolates were completely resistant. However, in2005, all but 10% of the isolates were sensitive to mefenoxam.From 2003 to 2005, SW Michigan vegetable growers eitheravoided or limited their use of mefenoxam because of its ineffectivenessin disease management due to fungicide resistance.Previously, a P. capsici field population from SW Michigan didnot revert from mefenoxam resistant to sensitive during a 2-yearrotation to non-host crops (21). The isolates collected in 2005from SW1 may have been introduced from a source differentfrom the nearby field or the number of sensitive isolates may beelevated due to the influx of a single asexual genotype. It has beennoted with another oomycete pathogen (Pythium aphanidermatum)that metalaxyl-resistant isolates declined in number aftermetalaxyl use was eliminated for at least 3 years. Although thesensitive isolates became more prevalent, they were not in numberslarge enough to allow the use of metalaxyl for diseasemanagement (45).P. capsici does not appear to be overwintering (asexually) in thewater sources. Fingerprints were not monomorphic across years atindividual monitoring sites and the pathogen was never detectedin the spring or early summer. In addition, the pathogen was neverdetected prior to mid-July or after late September, despite watermonitoring efforts which began in April and ended in late Octoberfor most sites in this study. Continued irrigation baiting in 2006has yielded P. capsici from cucumber fruit as early as 30 June(M. K. Hausbeck, unpublished data). There was no associationbetween P. capsici similarity groups and specific locations oryears. This may be due to the relatively small sample sets analyzedor could be due to long-distance movement of the pathogen viathe water sources. The genotypic diversity among the isolatesanalyzed underscores the importance of sexual recombination inthe life cycle of P. capsici and also shows that these water sourcesprovide an important, genetically diverse, reservoir of propagulesduring key vegetable production months. Use of surface water forirrigation of P. capsici-susceptible crops should be avoided toprevent further spread of the pathogen.ACKNOWLEDGMENTSThis study was funded by Michigan Specialty Crop Block Grant forPickling Cucumbers: Increase Marketability of Cucumbers for Picklingby Decreasing Fruit Rot Caused by Phytophthora and Pythium andProject GREEEN (a cooperative effort by plant-based commodities andbusinesses with Michigan State University Extension, the MichiganAgricultural Experiment Station, and the Michigan Department of Agriculture).We thank J. Woodworth and K. Cervantes of Michigan StateUniversity for technical support, and the Michigan grower cooperatorswho allowed us access to surface water sources and nearby fields.LITERATURE CITED1. Alconero, R., and Santiago, A. 1972. Characteristics of asexualsporulation in Phytophthora palmivora and Phytophthora parasiticanicotianae. Phytopathology 62:993-997.2. Babadoost, M., and Islam, S. Z. 2001. 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