Dynamics of Verticillium Species Microsclerotia in Field Soils in Response to Fumigation, Cropping Patterns, and Flooding

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1 Ecology nd Epidemiology Dynmics of Verticillium Species Microscleroti in Field Soils in Response to Fumigtion, Cropping Ptterns, nd Flooding Dyln P. G. Short, Germn Sndoy, Gry E. Vlld, Steven T. Koike, Chng-Lin Xio, Bo-Ming Wu, Surj Gurung, Ryn J. Hyes, nd Krishn V. Subbro First, seventh, nd ninth uthors: Deprtment of Plnt Pthology, nd second uthor: The Genome Center nd Deprtment of Plnt Sciences, University of Cliforni, Dvis, 1636 E Alisl St., Slins 93905; third uthor: Gulf Cost Reserch nd Eduction Center, University of Florid, IFAS, Wimum 33598; fourth uthor: University of Cliforni Coopertive Extension, 1432 Abbott Street, Slins 93901; fifth uthor: United Sttes Deprtment of Agriculture Agriculturl Reserch Service (USDA-ARS), Sn Joquin Vlley Agriculturl Sciences Center, 9611 S. Riverbend Ave, Prlier, CA 93648; sixth uthor: Deprtment of Plnt Pthology, Chin Agriculturl University, Beijing ; nd eighth uthor: USDA-ARS, 1636 E. Alisl St., Slins, CA Accepted for publiction 6 Jnury ABSTRACT Short, D. P. G., Sndoy, G., Vlld, G. E., Koike, S. T., Xio, C.-L., Wu, B.-M., Gurung, S., Hyes, R. J., nd Subbro, K. V Dynmics of Verticillium species microscleroti in field soils in response to fumigtion, cropping ptterns, nd flooding. Phytopthology 105: Verticillium dhlie is soilborne, economiclly significnt fungl plnt pthogen tht persists in the soil for up to 14 yers s melnized microscleroti (ms). Similrly, V. longisporum is very significnt production constrint on members of the fmily Brssiccee. Mngement of Verticillium wilt hs relied on methods tht reduce ms below cropspecific thresholds t which little or no disese develops. Methyl bromide, brod-spectrum biocide, hs been used s preplnt soil fumignt for over 50 yers to reduce V. dhlie ms. However, reductions in the number of ms in the verticl nd horizontl soil profiles nd the rte t which soil recoloniztion occurs hs not been studied. The dynmics of ms in soil before nd fter methyl bromide + chloropicrin fumigtion were followed over 3 yers in six 8-by-8-m sites in two fields. In seprte fields, the dynmics of ms in the 60-cm-deep verticl soil profile pre- nd postfumigtion with methyl bromide + chloropicrin followed by vrious cropping ptterns were studied over 4 yers. Finlly, ms densities were ssessed in six 8-by-8-m sites in seprte field prior to nd following nturl 6-week flood. Methyl bromide + chloripicrin significntly reduced but did not eliminte V. dhlie ms in either the verticl or horizontl soil profiles. In field studies, increses in ms were highly dependent upon the crop rottion pttern followed postfumigtion. In the verticl soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected t 60-cm depths. Six weeks of nturl flooding significntly reduced (on verge, pproximtely 65% in the totl vible counts of ms) but did not eliminte vible ms of V. longisporum. Mny soilborne fungi re cpble of surviving the dynmic soil microenvironment through the formtion of resilient resting structures, such s thick-wlled spores, melnized hyphe, nd scleroti (6). Verticillium dhlie is soilborne plnt-pthogenic fungus tht my persist in soil for up of 14 yers in the bsence of host due to its bility to form melnized microscleroti (ms) (27). Verticillium wilt disese cused by V. dhlie hs been n economiclly significnt problem on crops in the Slins Vlley for mny decdes but, on lettuce, it ws first discovered in 1995 (1). Strwberry, rtichoke, pepper, tomto, nd lettuce re significntly ffected by this disese in this re (5,9,11,17). On culiflower nd other members of the Brssiccee fmily, Verticillium wilt is cused by the relted species V. longisporum (13). The inoculum density (number of propgules unit _ 1 mss of soil) is significnt epidemiologicl fctor in Verticillium wilt (25). V. dhlie inoculum in soil my fluctute over time due to chemicl inputs (7), tillge (32), cropping ptterns (34), nd prolonged soil flooding (12,26). Mngement of Verticillium wilt is generlly directed t reducing the density of vible ms to levels below the threshold t which the disese no longer develops (4), becuse Verticillium wilt development in different crops is ssocited with vrying densities of vible ms (subsequent mentions of ms refer to vible ms). In severl crops, epidemics hve been correlted with reltively low Corresponding uthor: K. V. Subbro; E-mil ddress: kvsubbro@ucdvis.edu The Americn Phytopthologicl Society levels of ms. For exmple, 1 to 2 ms g _ 1 of soil resulted in 50% wilt incidence in strwberry (11), while culiflower required 10 ms g _ 1 of soil to cuse the sme level of disese incidence (33). Verticillium wilt in peppermint (30), potto (24,25), nd rtichoke (2) re lso ssocited with reltively low levels of inoculum in soil. In contrst, during surveys of lettuce fields with pproximtely 5% wilt incidence, estimtes of densities were 16 ms g _ 1 of soil on verge wheres, in fields with 80 nd 94% wilt incidence, densities were estimted t 932 nd 102 ms g _ 1 of soil, respectively (31), leding to the hypothesis tht Verticillium wilt of lettuce occurs only when densities re higher thn 100 ms g _ 1 of soil. Infested lettuce fields with up to 2,500 ms g _ 1 of soil hve been reported (1). Fumigtion to reduce V. dhlie ms in soil hs been widely prcticed for decdes in Cliforni (28). Methyl bromide (MeBr), brod-spectrum biocide commonly used with chloropicrin (7), while being highly efficcious, does not completely sterilize soil (7,15,28) nd offers only trnsient pthogen reduction. Becuse it is recognized s strtospheric ozone-depleting gent, it will finlly be phsed out from griculturl use in Cliforni by 2015 (28). Thus, it is importnt to quntify the effects of MeBr on pthogen distribution in fumigted soil nd how the postfumigtion cropping ptterns ffect this distribution. These results form the bsis for compring the efficcies of lterntive fumignts currently registered for griculturl use nd the potentil of future innovtive strtegies to reduce Verticillium wilt. A previous study (1) reported tht three infested fields with pproximtely 150, 40, nd 15 ms g _ 1 of soil treted with chloropicrin-telone, chloropicrin, nd MeBr, respectively, showed 638 PHYTOPATHOLOGY

2 reductions in densities to less thn 5 ms g _ 1 of soil following fumigtion. All three fields experienced significnt rebound in pthogen popultion growth when lettuce crops were grown 1 yer postfumigtion (1). However, multiyer quntittive dt bsed on intensive smpling is needed to investigte the limittions of chemicl fumignts nd the effects of cropping ptterns on chnges in V. dhlie recoloniztion of soil postfumigtion. A limited number of multiyer studies hve reported popultion fluctutions in V. dhlie in response to fumigtion nd cropping ptterns (21). For exmple, previous study hs reported tht fllow fields retin ms levels t ner constnt levels (4) nd my be expected to decrese only slightly over time, wheres lettuce crops my cuse ms levels to increse (1), consistent with reports of vrible ms production on different crops (8). Preliminry dt hs recorded fluctutions from 10 ms g _ 1 of soil postfumigtion to >600 ms following two successive crops of lettuce (1). Conversely, there is evidence tht broccoli cultivtion suppresses the growth of V. dhlie in fields nd ctully leds to decrese in soil ms levels over time (21). In ddition to chemicl inputs, wter submergence of field soils cn gretly lter microbil soil communities, primrily by reducing vilble oxygen (22,23), nd hs been investigted s culturl control for mnging pthogens. For exmple, flooding hs been studied for its bility to destroy Sclerotini (16,19) nd Leptospheri (22) reservoirs in fields. Previous investigtions hve reveled tht cultivtion of rice, millet, nd sorghum under flooding cn reduce V. dhlie in soil nd reduce Verticillium wilt on subsequent crops (4,9). Furthermore, the effectiveness of flooding s culturl prctice to reduce or eliminte V. dhlie from soil my be dependent on temperture (20). Flooding over 12-week period successfully reduced V. dhlie to below detectble limits (26), nd 6-week flooding time hs been estimted for successfully eliminting vible V. dhlie propgules in soil (18), bsed on limited smll-scle experiments. Thus, s strtegy to reduce vible ms, flooding hs promise s pthogen mngement technique if wter resources nd their use re unlimited. Given the long-term persistence of V. dhlie in soil environments nd the inevitble phse-out of MeBr, it is criticl to understnd how V. dhlie popultions vry over time so tht the efficcy of lterntive tretments or culturl prctices, including flooding, cn be sufficiently evluted reltive to MeBr. Knowledge of soilborne fungl dynmics, especilly the rebound of soilborne pthogen popultions, would lso be useful to develop epidemiologicl models for soilborne diseses nd mke informed mngement decisions. Currently, multiyer quntifictions of soil densities of V. dhlie re lcking, prticulrly in response to chemicl fumignts, cropping ptterns, nd cross different depths within the soil profile. Therefore, the objectives of the current study were to (i) determine the effects of fumigtion on ms density in the horizontl soil profile, (ii) determine the dynmics of ms in the verticl soil profile postfumigtion nd under two cropping systems (fumigtion followed by strwberry nd continuous lettuce versus fumigtion followed by strwberry nd lettuce interspersed with culiflower), nd (iii) ssess the effects of 6-week nturl flooding on ms densities within two different fields. MATERIALS AND METHODS Dynmics of ms densities in the horizontl soil profile. These studies were conducted in fields locted in Wtsonville, CA nd both developed pproximtely 90% wilt incidence in 2004 on lettuce crops. Becuse of the ner-totl loss of lettuce crops, the grower decided to fumigte the fields with MeBr + chloropicrin (66: 33) t rte of 393 kg h _ 1. Soils t ll the experimentl sites ws n Elder sndy lom (corse-lomy, mixed, thermic, Cumulic Hploxeroll) with 62% snd, 26% silt, 0.6% orgnic mtter, nd ph of 6.5. Soil smples were collected prior to fumigtion in October to determine the distribution of ms in the horizontl profile in ech field. Within ech field, three 8-by-8-m grids of 64 contiguous qudrts (1 by 1 m), seprted by 75 m, were demrcted nd site loctions were recorded using GPS unit (Grmin, Schffhusen, Switzerlnd). At ech site, 64 prefumigtion soil smples were collected in 2004 followed by n dditionl 64 postfumigtion smples in 2005, 2006, nd 2007 from the top 7 to 10 cm using 6.25-cm-dimeter soil probe. Soil smples were ssyed to determine the number of ms in ech soil smple, s described below. Following fumigtion, the cropping pttern in field 1 ws strwberry romine lettuce crisphed lettuce nd in field 2, the cropping sequence ws strwberry cbbge crisphed lettuce. Determintion of V. dhlie ms in soil smples. The number of V. dhlie ms in ech smple ws ssyed by impction plting of pulverized soil using the modified Anderson smpler technique (3), s previously described (32). Briefly, soil smples were plced in pper bgs nd ir dried for 4 to 6 weeks on greenhouse bench. Soil ws pulverized with mortr nd pestle nd sifted through mm sieve. Soil (10 g) ws plced in snp-cp vil nd mixed with 2.5 ml of DL-methionine solution (7.5 mg ml _ 1 ) nd incubted t 30 C for 1 week, then opened nd ir dried for 1 week t 22 to 24 C. With the Anderson smpler, 0.5 g of pulverized soil ws distributed on six petri pltes contining NP-10 medi (3,13) nd incubted t mbient indoor temperture in the drk. The numbers of V. dhlie colonies were counted 3 to 4 weeks fter plting using stereomicroscope. Counts from the six pltes were combined s the number of colonies of V. dhlie per 0.5 g of soil nd expressed s the number of ms g _ 1 of dry soil. The dt were nlyzed using SAS (version 9.3; SAS Institute, Cry, NC) to test the null hypothesis tht the increse or decrese in ms densities over the yers ws different from zero. An unstructured covrince model ws used nd the covrince mtrix ws modeled using replictes nested within block nd field. The 2004 ms dt were used s bseline nd included in the model s covrible. Ech site ws considered block (three blocks per field) with 64 smpling points or replictions (Figs. 1 nd 2). Bseline, fields, time, nd the field time interction were considered fixed fctors, while blocks nested within fields were treted s rndom effects. The F vlue ws used to detect significnt effects in the nlysis of vrince (ANOVA) model, P vlues 0.05 were considered significnt. Lest squre mens (LSM) were clculted for ech field in ech yer nd compred between fields using the P vlues. Two LSM were considered different when P ws t lest Verticl distribution of ms nd soil recoloniztion following fumigtion nd cropping sequences. In fields locted pproximtely 5 km wy from those described bove in 2002, lettuce crops lso developed Verticillium wilt nd resulted in ner-totl yield loss. Becuse different cropping ptterns were plnned in these fields following fumigtion (Tble 1), fields were designted blocks 2, 3, 4, nd 2 L to fcilitte esier identifiction. Fields in both loctions were owned by the sme grower, who decided to fumigte these fields lso with MeBr + chloropicrin t rte of 393 kg h _ 1. Blocks 2, 2 L, nd 3 were fumigted in the fll of 2002 while block 4 ws left s nonfumigted control. Vrious crop rottions were followed in the blocks, s follows: block 2 = strwberry (two sesons), fllow field, rdicchio, fllow field, crisphed lettuce, romine lettuce; block 3 = strwberry (two sesons), fllow field, rdicchio, fllow field, red cbbge, romine lettuce; block 4 = strwberry (two sesons) fllow field, crisphed lettuce (three sesons), nd culiflower (Tble 1); however, block 2 L ws low-lying prt of the field tht ws too wet to plnt crops nd, thus, ws left fllow for the durtion of this study. Ech spring from 2002 (prefumigtion smples) to 2005 (postfumigtion smples to determine soil recoloniztion), 48 soil smples (12 smples from four sites) were collected in ech block in 5-cm increments to depth of 60 cm using 6.25-cm-dimeter soil probe. Smpling sites were geo-referenced to obtin yerly smples in the vicinity of these loctions. The number of ms in ech soil smple ws determined using the technique described bove. Vol. 105, No. 5,

3 Fig. 1. Verticillium dhlie microscleroti g _ 1 of soil estimtes for field 1 over the 4-yer study period ner Wtsonville, CA. Fig. 2. Verticillium dhlie microscleroti g _ 1 of soil estimtes for field 2 over the 4-yer study period ner Wtsonville, CA. 640 PHYTOPATHOLOGY

4 The differences in the levels of ms in different blocks nd depths over time were nlyzed using repeted mesures ANOVA in PROC MIXED in SAS (version 9.3; SAS Institute) following Littell et l. (2006) (14). An utoregressive order 1 covrince structure ws used. The 2002 ms dt were used s bseline nd fit in the model s covrible. In the ANOVA, bseline, blocks, depths, nd times were considered fixed effects. LSM were clculted for ech depth yer block tretment combintion. When interctions were significnt, differences in the min tretment levels were tested for significnce using the SAS slice option t P Three types of comprison were mde. The first compred the differences between yers within ech block nd soil depth. In the second, differences mong soil depths within ech block nd yer were compred. The third comprison ws mde between blocks within ech depth nd yer. Becuse the cropping sequences influenced ms densities within ech block, the recoloniztion of soil postfumigtion by V. dhlie ws directly relted to the ms densities within ech block. Chnges in V. longisporum ms density in response to flooding. Three study sites were demrcted in ech of two fields infested with V. dhlie, designted A nd B. Ech site ws divided into 64 contiguous 1-by-1-m qudrts. The cropping pttern of these fields t the time of study consisted of culiflower in nnul rottion with lettuce. In the Slins Vlley, CA, intense flooding occurred in Jnury nd Mrch 1995 s result of significnt winter storms, which left stnding wter on sturted fields for 4 weeks. Significnt precipittion lso occurred subsequent to the pek periods of winter storms. After stnding wter hd drined, fields remined too wet for tillge opertions for n dditionl 3 to 4 weeks. Therefore, the fields were under flooded conditions for t lest 6 weeks. Soil cores were collected to depth of 15 cm with 2.5-cm-dimeter soil probe preflooding (31) nd postflooding (n dditionl 6 weeks fter the flooding period ended) from the sme qudrts. The number of V. dhlie ms in ech smple ws ssyed s described bove. Differences between ms densities preflooding nd postflooding were computed using n ANOVA with the MIXED procedure of SAS (version 9.3; SAS Institute). Flooding sttus (pre nd post), fields, nd flood field were considered fixed fctors, wheres the interction of block (flooding field) ws considered rndom fctor. Mens (LSM) were computed nd were considered significntly different when P ws t lest RESULTS Densities of ms in response to fumigtion in the horizontl soil profile. Both lettuce fields used in this study hd up to 90% wilt incidence (in the re where the smpling loctions were demrcted) in the lettuce crops in 2004 nd contined high but unequl mounts of ms prior to fumigtion. Field 1 hd n verge of 46 ms g _ 1 of soil nd field 2 contined men of 38 ms g _ 1 of soil. The use of the 2004 dt s bseline ws done to djust the nlysis for the unblnced ms levels t the beginning of the experiment (Tble 2), nd this ws sttisticlly significnt (P = TABLE 1. Postfumigtion cropping history in the four blocks of field infested by Verticillium dhlie t the beginning of the verticl soil profile study Block Spring* Fll Spring* Fll Spring* Fll Spring* Fll 2 CL F-St St _ Rdi _ CL RoL 2 L CL F 3 CL F-St St _ Rdi _ RCb RoL 4 CL NF-St St _ CL CL CL Cu Asterisks (*) indicte smpling times, _ = fllow field, F = fumigtion, NF = nonfumigtion, CL = crisphed lettuce, RoL = romine lettuce, Rdi = rdicchio, St = strwberry, cu = culiflower, GCb = green cbbge, nd Rcb = Red cbbge ) (Tble 2). In the ANOVA, differences between fields were not significnt (P = ), wheres time nd the time field interction were highly significnt (P > ) (Tble 2). In both fields, fumigtion reduced but did not completely eliminte detectble levels of V. dhlie ms (Figs. 1 nd 2). After fumigtion, the levels of ms in both fields ws reduced to pproximtely 3 ms g _ 1 of soil (Tble 3). Both fields were plnted to strwberry following fumigtion nd there ws smll increse in the number of ms to pproximtely 5 ms g _ 1 of soil in 2006 (Tble 3). The cropping ptterns of the two fields up until then were identicl nd the men number of ms g _ 1 of soil ws not significntly different in 2005 (P=0.9680) or 2006 (P = ) (Tble 3). However, in field 1, romine lettuce ws cultivted in 2007 nd verge inoculum density incresed to 75 ms g _ 1 of soil. In field 2, cbbge crop ws grown insted nd only 6 ms g _ 1 of soil were found (Tble 3). The men number of ms per grm of soil in the two fields in 2007 ws significntly different (P = ). The inoculum distribution in the horizontl soil profile over time in field 1 ws pproximted by the distribution of ms prior to fumigtion in ll three sites, lthough some slight differences in the redistribution of soil ms ws pprent, which could be ttributed to soil tillge prctices (Fig. 1). In contrst, the distribution of ms postfumigtion ws mintined for t lest 2 yers following fumigtion in field 2 (Fig. 2). Verticl distribution of ms pre- nd postfumigtion. The inoculum distribution in the verticl soil profile ws evluted in fields with history of Verticillium wilt in lettuce crops. Prior to fumigtion in 2002, the levels of ms were different in ech block regrdless of the depth (dt not shown). In ddition, the mounts of ms in 2002 prior to fumigtion were higher thn in subsequent yers (2003, 2004, nd 2005) (dt not shown). Therefore, the 2002 dt were used s bseline in the model, which ws significntly higher thn in ll other yers (P < ). Blocks, depths, time, nd their interctions were lso significnt (P < ) in the ANOVA (Tble 4). LSM of ms differed mong yers in blocks 2, 3, nd 4 t depths of 5 cm (Tble 5). Differences between yers were detected t greter depths in block 2 (5, 10, 15, 20, 25, nd 35 cm) nd block 3 (5, 10, 15, 20, 25, nd 30 cm) (Fig. 3). Overll, the highest mounts of ms were found in 2004 in blocks 2 nd 3 (Tble 5). There were no differences in the numbers of ms t ny soil depth in block 2 L (Tble 5). The nonfumigted block 4 initilly (2002) hd lower numbers of ms compred with blocks 2 nd 3 (dt not shown) nd TABLE 2. Repeted mesures nlysis of vrince for the level of microscleroti in two different field sites in Slins, CA with different cropping histories Effect Num DF Den DF F vlue P > F b Bseline Fields Times < Times fields < Bseline dt were microscleroti from 2004 nd times represent the response of the microscleroti over the yers (2005, 2006, nd 2007). b Differences significnt t P TABLE 3. Lest squre mens of the level of microscleroti in three different yers (2005 to 2007) t two different field sites in Wtsonville, CA with different cropping histories Yers Fields b Field Field P vlue Mens re the verge of 192 smpling points ech yer nd field. b Both fields were fumigted in Field 1 ws plnted with strwberry (2005), crisphed lettuce (2006), nd romine lettuce (2007). Field 2 ws plnted with strwberry (2005), crisphed lettuce (2006), nd cbbge (2007). Vol. 105, No. 5,

5 generlly did not hve significnt chnges in ms, with the exception of n increse t 5 cm in 2005 (Tble 5). At incresing soil depths, ms decresed in blocks 2 nd 3, beginning in 2004 (Tble 5). The reductions were significnt in ll yers for block 2 nd in 2003 nd 2004 for block 3. Differences between blocks were observed nd the highest ms were generlly observed in blocks 2 (P in 2003, 2004, nd 2005) nd 3 (P in 2003 nd 2004); no significnt differences were observed in block 3 in 2005 (P = ) or blocks 2L(P = 1, , nd 1 in 2003, 2004, nd 2005, respectively) nd block 4 (P = , , nd in 2003, 2004, nd 2005, respectively). Differences between blocks were detected t depth of 5 cm in ll yers. Significnt differences between blocks were detected down to 35 nd 45 cm in 2004 (Tble 6) nd t 5, 10, 15, nd 25 cm in 2005 (Tble 6). Chnges in V. longisporum ms density nd field distribution in response to flooding. Preflooding smpling conducted cross fields A nd B in six different blocks indicted tht ms distributions were not equl within ech field. Field A hd higher densities of ms thn field B (Fig. 4). The postflooding soil distributions of ms were lso not equl in both fields. The nlysis of vrince showed significnt differences in the levels of ms due to flooding (Tble 7). Differences between fields were not significnt, wheres the flooding field interction ws significnt (P = ). A higher reduction of ms ws detected in field A (63%) thn in field B (14%); ms were detected in ll six blocks fter flooding (Fig. 4). The level of ms ws reduced postflooding but not eliminted in either field. The LSM of the levels of ms from both fields, bsed on 384 smpling points from both field sites, were ms g _ 1 of soil before flooding nd 7.32 ms g _ 1 of soil fter flooding, with P vlue of DISCUSSION Understnding the dynmics of fungl popultion fluctutions in soil in response to biotic fctors, including chemicl inputs, is TABLE 4. Repeted mesures nlysis of vrince for the level of microscleroti smpled in four block t 12 depths between 5 nd 60 cm between 2003 nd 2005 Effect Num DF Den DF F vlue P > F Bseline < Block b < Depth < Time Depth block < Time block < Time depth < Time depth block Dt from 2002 were used s bseline, which ws introduced into the model s covrible. b Three blocks were fumigted blocks while one ws not fumigted. importnt for sustinble mngement of reclcitrnt soilborne pthogens such s V. dhlie. Knowledge of how fungl inoculum chnges over time is importnt to estimte the potentil disese pressure, bsed on which, cropping decisions cn be mde. In this study, we determined the effects of fumigtion on the ms densities in both the horizontl nd verticl soil profiles nd lso determined how these densities re ffected by cropping prctices nd nturl flooding event. In the first experiment, soils from two fields were smpled prior to fumigtion nd during subsequent sesons to monitor nd mp V. dhlie ms levels in the soil. As expected, drmtic decreses in ms densities were observed in the three sites within both fields following fumigtion with MeBr nd chloropicrin. A yer fter fumigtion, the mjority of smples contined no detectble ms. Over time, the counts of vible ms incresed, suggesting tht the V. dhlie popultion hd reproduced on subsequent crops. Up until the third smpling, the two fields followed identicl cropping ptterns but, fter the third smpling, field 1 ws plnted with romine lettuce wheres field 2 ws plnted with culiflower. This difference in single crop between the third nd fourth smpling times ppers to hve plyed criticl role in the increse of V. dhlie within field soil becuse V. dhlie is not pthogenic on culiflower (32). In ll three sites in field 2 before nd fter culiflower, ms densities were not significntly different. In contrst, ms densities in ll three sites in field 1 showed significnt increses over soils smpled prior to the romine lettuce crop. Within the lettuce field, there ws strong evidence tht soil levels of ms hd incresed bck to the prefumigtion levels. We ttribute these differences to the cropping pttern itself, nd deduce tht lettuce enbled the fungus to rpidly reestblish within the soil, reltive to culiflower. Culiflower nd lettuce both develop Verticillium wilt but the cusl gents in the two crops re different. Although Verticillium wilt in lettuce is cused by V. dhlie, in culiflower it is cused by the sister species V. longisporum (13), nd the two species re not cross-pthogenic on the two crops (32). Thus, even if one species is present in the soil, plnting the nonhost species does not ugment the soil inoculum levels nd, thus, the results of this experiment strongly suggest tht production of ms in lettuce by V. dhlie ws significntly greter thn on culiflower. These results indicte tht fumigtion with MeBr + chloropicrin significntly reduced but did not eliminte ms below the detection limit of <2 CFU/g of soil. We estimted tht multiple smples in different replictes nd experiments contined between 50 nd 100 ms g _ 1 of soil following fumigtion, which, ccording to models of V. dhlie epidemiology, would be over the predicted threshold for disese development in ll crops studied to dte, except lettuce (31). There is evidence tht sites with high initil levels of ms experience fster rebounds of ms in soil (31). Furthermore, it ppers tht some crops, especilly lettuce, ply n importnt role in providing conducive environments for the TABLE 5. Lest squre mens of the level of microscleroti in three different yers (2003 to 2005) fter fumigtion, in blocks Block 2 L Block 2 Block 3 Block 4 Depths P vlue P vlue P vlue P vlue < < < Block 2 L = not fumigted, blocks 2 nd 3 = fumigted, nd block 4 = controls. P vlues re the effect of yers within ech soil depth for ech block. 642 PHYTOPATHOLOGY

6 fungus to reproduce rpidly nd rech epidemiologiclly relevnt levels under fvorble conditions. The modes of ction of MeBr + chloropicrin re not precisely known. Therefore, it remins to be seen whether residul V. dhlie in fumigted fields re somehow phenotypiclly more resilient to exposure to these chemicls or re the result of inefficient vpor penetrtion throughout the vst soil micropore mtrix. We hve demonstrted tht pockets of V. dhlie reservoirs my remin in field soil following fumigtion, nd tht these events represent popultion bottlenecks which drsticlly reduce V. dhlie popultion genetic diversity. This is supported by the fct tht V. dhlie popultions in the Slins Vlley re dominted by only few widespred genotypes which hve been prevlent since 1995 (10). In the second experiment, we explored the dynmics of V. dhlie ms densities in the verticl soil profile s influenced by fumigtion nd differences in postfumigtion cropping ptterns. The reltionship between Verticillium wilt incidence nd densities of ms follows threshold of pproximtely 150 ms g _ 1 of soil for lettuce crops to develop significnt levels of wilt (31). The prefumigtion densities of ms in block 2 ws in line with this inoculum threshold, becuse 90% incidence ws correlted with n verge of 493 ms g _ 1 of soil. In contrst, block 2 L, which hd levels of Verticillium wilt incidence similr to those in block 2, contined less thn 10 ms g _ 1 of soil. Block 2 L ws locted in lower prt of the field with stgnnt wter, nd this my hve reduced the number of ms in this block. The wilt incidence in block 3, estimted to be between 10 nd 15%, ws correlted with 66 ms g _ 1 of soil wheres block 4, which hd no wilt, contined pproximtely 28 ms g _ 1 of soil, gin vlidting the inoculum threshold required for high incidence of Verticillium wilt in lettuce (31). These studies did not tke into ccount the pthogenic rces present in the soil. Preliminry evidence from field inocultion studies suggest tht the fecundity of rce 1 reference strin V. dhlie Ls16 is much higher thn tht of rce 2 reference strin V. dhlie Ls17 when inoculted into Slins lettuce. Soils from field plnted with Ls16-inoculted plnts contined much higher vible ms densities following seson of crop growth compred with the neighboring field plnted with Ls17-inoculted plnts (K. V. Subbro, unpublished dt). Further studies re needed to determine the vribility in inoculum production nd ssocited disese incidence mong strins of V. dhlie belonging to rces 1 nd 2. In blocks 2 nd 3, the cropping ptterns were similr, except during the spring of 2005, when crisphed lettuce ws grown in block 2 nd red cbbge ws grown in block 3 insted. The combintion of higher wilt incidence prior to fumigtion in block 2, consistently higher soil inoculum densities, nd plnting of successive susceptible crops influenced the increse in V. dhlie ms cross ll depths reltive to block 3. In contrst, in block 4, lck of disese in 2002 nd low levels of ms cross ll depths were kept low through 2005 despite the plnting of susceptible crops throughout the study, except in the fll of As demonstrted TABLE 6. Probbility vlues from the slice option in SAS for the effect of block on microscleroti within ech depth nd yer Yers Depths < < < < < < < < Fig. 3. Verticillium dhlie microscleroti g _ 1 of soil estimtes from 5 to 60 cm within blocks 2, 3, 4, nd 2 L. Stndrd errors re shown with error brs. Vol. 105, No. 5,

7 in the postfumigtion increse in V. dhlie ms in the horizontl soil profile, the cropping ptterns criticlly influenced the dynmics of V. dhlie ms in the verticl soil profile s well. The presence of inoculum t depths where plnt roots grow is one of the most epidemiologiclly relevnt fctors, nd different crops my be ffected by pthogens present t different depths owing to differences in plnt root rchitecture. Generlly speking, when the dt from ll four blocks were combined from the second experiment, the top 5 cm consistently contined the highest ms densities, nd the top 5 to 15 or 20 cm likewise contined significntly higher numbers of ms thn deeper depths. The presence of greter numbers of ms ner the soil surfce is presumbly due to the growth nd reproduction of V. dhlie on plnt roots nd on debris in the top 25 cm of the soil profile. The root rchitecture of lettuce, in prticulr (15,16), my represent highly conducive niche for reproduction mong the diverse hosts of V. dhlie becuse much of the root biomss (tproot nd the extensive dventitious root system) is locted within the rised beds. Even t 60 cm of depth, ms were consistently detected in ll the blocks, which suggests tht tillge of soil my mechniclly disperse ms from soil depths ner the surfce to deeper depths. Soil tillge lso plys role in moving ms cross fields, becuse we observed reduction in ms in multiple sections of the verticl soil profile within nonfumigted control site. This is ttributble to the dispersl of ms from the smpling site vi tillge, s well s nturl locl-level dispersl. Tken with the observtion tht rre ptches of residul ms remin in fields, the movement of ms vi tillge my llow V. dhlie popultions to reestblish more quickly, becuse more ms re spred to res plnted with host crops. This observtion is in-line with recent popultion genetics nlyses of V. dhlie in the Slins Vlley, CA, which reveled persistent, widespred, highly clonl popultions in commercil fields (10), consistent with the ide of clonets rising from point source reservoirs of reclcitrnt genotypes. Although fumigtion my significntly reduce most V. dhlie within infested field sites, it my lso drive popultion bottlenecks nd indvertently select for reclcitrnt genotypes to subsequently infect nd dominte the rhizosphere. Verticillium hs been reported to survive incubtion under nerobic conditions (8), which would theoreticlly llow survivl of ms beyond the initil period of oxygen depletion tht occurs in soil submergence (23). A previous report found tht 6 weeks of flooding were required to decrese ms densities in soil (26), which fell from 50 to 60 to 0 to 10 ms g _ 1 of soil over the course of 17 weeks. Bsed on these observtions, Pullmn nd DeVy (26) hypothesized tht Verticillium ms my withstnd flooding conditions for up to 6 weeks, due to their resilient, multicellulr nture. In the current study, fter 6-week period of stgnnt flood wters, three sites experienced, on verge, pproximtely 65% reduction in counts of vible ms. In severl smpled qudrts in five of the six sites, ms were not detected. However, the mjority of qudrts still contined 6 to 45 ms g _ 1 of soil. Temperture effects on the efficcy of flooding in reducing Verticillium ms hve lso been reported (20,26); in the current study, TABLE 7. Anlysis of vrince for the number of microscleroti before nd fter flooding in two different field sites in Slins, CA during 1995 Effect Num DF Den DF F vlue P > F Flooding Fields Flooding field Rep (block) Significnt differences (P 0.05). Fig. 4. Verticillium longisporum microscleroti g _ 1 of soil estimtes within field in Slins, CA prior to nd following 6 weeks of nturl flooding. 644 PHYTOPATHOLOGY

8 flooding in the Slins Vlley, which experiences cool Mediterrnen climte, occurred throughout spring Thus, results from this study only prtilly support previous findings on the efficcy of flood tretments to eliminte this importnt soilborne pthogen. In conclusion, quntifying soil levels of ms before nd fter fumigtion with MeBr nd chloropicrin is importnt to hve bseline for comprtive studies on lterntive soil tretments nd evlutions of their efficcy. This study provides quntittive estimtes of the density of residul Verticillium ms which survive in soil postfumigtion nd differing cropping sequences, nd sturtion with flood wters. ACKNOWLEDGMENTS Mention of trde nmes or commercil products in this rticle is solely for the purpose of providing specific informtion nd does not imply recommendtions or endorsement by the U.S. Deprtment of Agriculture. USDA is n equl opportunity provider nd employer. LITERATURE CITED 1. Atllh, Z. K., Hyes, R. J., nd Subbro, K. V Fifteen yers of Verticillium wilt of lettuce in Americ s sld bowl: A tle of immigrtion, subjugtion, nd btement. Plnt Dis. 95: Berbegl, M., Orteg, A., Grcí-Jiménez, J., nd Armengol, J Inoculum density-disese development reltionship in Verticillium wilt of rtichoke cused by Verticillium dhlie. Plnt Dis. 91: Butterfield, E. J., nd DeVy, J. E Ressessment of soil ssys for Verticillium dhlie. Phytopthology 67: Butterfield,E.J.,DeVy,J.E.,ndGrber,R.H.1978.Theinfluenceof severl crop sequences on the incidence of Verticillium wilt of cotton nd on the popultions of Verticillium dhlie in field soil. Phytopthology 68: Cirulli, M., Bubici, G., Amenduni, M., Armengol, J., Berbegl, M., Jiménez-Gsco, M. M., nd Jiménez-Díz, R. M Verticillium wilt: A thret to rtichoke production. Plnt Dis. 94: Coley-Smith, J. R., nd Cooke, R. C Survivl nd germintion of fungl scleroti. Annu. Rev. Phytopthol. 9: Duniwy, J. M Sttus of chemicl lterntives to methyl bromide for pre-plnt fumigtion of soil. Phytopthology 92: Ebihr, Y., nd Uemtsu, S Survivl of strwberry-pthogenic fungi Fusrium oxysporum f. sp. frgrie, Phytophthor cctorum nd Verticillium dhlie under nerobic conditions. J. Gen. Plnt Pthol. 80: Ebihr, Y., Uemtsu, S., nd Nomiy, S Control of Verticillium dhlie t strwberry nursery by pddy-uplnd rottion. J. Gen. Plnt Pthol. 76: Gurung, S., Short, D. P. G., Atllh, Z. K., nd Subbro, K. V Clonl expnsion of Verticillium dhlie in lettuce. Phytopthology 104: Hrris, D. C., nd Yng, J. R The reltionship between the mount of Verticillium dhlie in soil nd the incidence of strwberry wilt s bsis for disese risk prediction. Plnt Pthol. 45: Ionnou, N., Schneider, R. W., nd Grogn, R. G Effect of flooding on the soil gs composition nd the production of microscleroti by Verticillium dhlie in the field. Phytopthology 67: Koike, S. T., Subbro, K. V., Dvis, R. M., Gordon, T. R., nd Hubbrd, J. C Verticillium wilt of culiflower in Cliforni. Plnt Dis. 78: Littell, R. C., Milliken, G. A., Stroup, W. W., Wolfinger, R. D., nd Schbenberger, O SAS for Mixed Models. SAS Institute, Cry, NC. 15. Mrtin, F. N., nd Bull, C. T Biologicl pproches for control of root pthogens of strwberry. Phytopthology 92: Mtheron, M. E., nd Porchs, M Influence of soil temperture nd moisture on eruptive germintion nd vibility of scleroti of Sclerotini minor nd S. sclerotiorum. Plnt Dis. 89: McCin, A. H., Rbe, R. D., nd Wilhelm, S Plnts Resistnt to or Susceptible to Verticillium Wilt. University of Cliforni Leflet Menzies, J. D Effect of nerobic fermenttion in soil on survivl of scleroti of Verticillium dhlie. (Abstr.) Phytopthology 52: Moore, W. D Flooding s mens of destroying the scleroti of Sclerotini sclerotiorum. Phytopthology 39: Ndkvukren, M. J The effect of soil moisture nd temperture on the survivl of Verticillium microscleroti. PhD dissert. Oregon Stte University, Corvllis. 21. Njoroge, S. M., Kbir, Z., Mrtin, F. N., Koike, S. T., nd Subbro, K. V Comprison of crop rottion for Verticillium wilt mngement nd effect on Pythium species in conventionl nd orgnic strwberry production. Plnt Dis. 93: Peluol, C., Fernndo, W. G. D., Huvenrs, C., Kutcher, H. R., Lhlli, R., nd Peng, G Effect of flooding on the survivl of Leptospheri spp. in cnol stubble. Plnt Pthol. 62: Ponnmperum, F The Chemistry of Submerged Soils, Vol. 24. Acdemic Press, New York. 24. Powelson, M. L., nd Rowe, R. C Biology nd mngement of erly dying of pottoes. Annu. Rev. Phytopthol. 31: Powelson, R Significnce of Popultion Level of Verticillium in soil. University Cliforni Press, Berkeley, CA. 26. Pullmn, G. S., nd DeVy, J. E Effect of soil flooding nd pddy rice culture on the survivl of Verticillium dhlie nd incidence of Verticillium wilt in cotton. Phytopthology 71: Schnthorst, W Life cycle nd epidemiology of Verticillium. Pges in: Fungl Wilt Diseses of Plnts. M. E. Mce, A. A. Bell, nd C. H. Beckmn, eds. Acdemic Press, New York. 28. Subbro, K. V Introduction. Phytopthology 92: Subbro, K. V., Vlld, G. E., Qin, Q., Koike, S. T., nd Hyes, R. J Biology nd epidemiology of Verticillium wilt of lettuce. Pges in: Cliforni Lettuce Res. Bord Annu. Rep. Cliforni Lettuce Reserch Bord, Slins, CA. 30. Tjmos, E. C., Rowe, R. C., Hele, J. B., nd Frvel, D. R Advnces in Verticillium Reserch nd Disese Mngement. The Americn Phytopthologicl Society, St. Pul, MN. 31. Wu, B. M., nd Subbro, K. V A model for multi-sesonl spred of Verticillium wilt of lettuce. Phytopthology 104: Xio, C. L., Ho, J. J., nd Subbro, K. V Sptil ptterns of microscleroti of Verticillium dhlie in soil nd Verticillium wilt of culiflower. Phytopthology 87: Xio, C. L., nd Subbro, K. V Reltionships between Verticillium dhlie inoculum density nd wilt incidence, severity, nd growth of culiflower. Phytopthology 88: Xio, C. L., Subbro, K. V., Schulbch, K., nd Koike, S. T Effects of crop rottion nd irrigtion on Verticillium dhlie microscleroti in soil nd wilt in culiflower. Phytopthology 88: Vol. 105, No. 5,