Identification, Distribution, and Aggressiveness of Spring Dead Spot ...

ponsoredResearch You Can elseIdentification, Distribution,andAggressiveness of Spring Dead SpotPathogens of BermudagrassUnderstanding what organisms cause thedisease and their geographic distribution.BY NED TISSERA"T, FANNY IRIARTE, HENRY WETZEL III, JACK FRY, AND DENNIS MARTINSpring dead spot (SDS) is a destructivedisease of common bermudagrass(Cynodon dactylon) andbermudagrass hybrids (C. dactylon XC. transvaalensis) throughout the northernrange of its adaptation in the UnitedStates. It may occur on bermudagrassfairways and putting surfaces of all ages,although it typically appears 3-4 yearsafter the turf has been established. Thedisease results in the formation ofcircular or arc-shaped patches of deadturf in early spring as bermudagrassbreaks winter dormancy.The dead patches, which are slightlydepressed and straw-colored, may rangein size from several inches to severalfeet in diameter. Roots and stolons ofaffected plants are dark brown to blackand are severely rotted. In some regions,such as Australia and California, patchesmay be visible on slowly growing,butnot dormant bermudagrass followingwet, cold weather. Bermudagrass slowlyfills in the bare areas during the growingseason, and by late summer there maybe little or no evidence of the disease.Dead patches reappear the followingspring in the same locations.IDENTIFICATIONOF SDS PATHOGENSIdentifying the cause(s) ofSDS hasbeen an elusive and frustrating process.We now know that three closely relatedOphiosphaerella korrae ascospores are initiallygrouped together in a sac-like structure calledan ascus. Individual ascospores are light brown,long, and cylindrical with multiple septations.The three Ophiosphaerella species that causespring dead spot can be differentiated by sporelength.root-rotting fungi called Ophiosphaerellakorrae (also called Leptosphaeria korrae),O. herpotricha, and O. narmari cause SDS.It is important to determine whichOphiosphaerella species is the cause ofSDS at a specific location because thesepathogens may differ in seasonal development,sensitivity to fungicides, andbermuda-aggressiveness to individualgrass cultivars.Unfortunately, these fungi are noteasily distinguished in the field becausethey cause identical symptoms. Theycan be differentiated in the laboratoryby spore (ascospore) length, with thoseof O. herpotricha being the longest, O.korrae intermediate, and O. narmari theshortest. However, these fungi seldomproduce fruiting structures (calledpseudothecia) containing ascospores ondiseased bermudagrass stolons andcrowns in the field, and only certainisolates can be induced to producethem in culture. Therefore, spore morphologycannot routinely be used todistinguish these fungi.To facilitate rapid identification,weemployed a polymerase chain reactiontechnique (PCR) to selectively amplifysmall segments of DNA from the threeNinety days after inoculation with Ophiosphaerellakorrae (middle) and O. herpotricha(right). the Arizona common bermudagrassroots showed discoloration and rotting. Rootson the left were not inoculated.MARCH-APRIL 2004 15

Ophiosphaerella species. By using speciesspecificoligonucleotide primers, DNAcan be differentially amplified followingDNA extraction from fungal culturesor diseased tissue. For example, if a PCRreaction is run using primers specificfor o. narmari, only DNA of this fungusand not others, including other Ophiosphaerellaspecies, will be amplified.Thus, the use of PCR primers specificto the three SDS pathogens can be usedto determine which fungus is present ina diseased root. Amplified DNA can bedetected in several ways, but commonlyit is stained, loaded into an agarose gelplaced in a buffer solution, and thensubjected to an electrical current. As theamplified DNA migrates through thegel, it can be visualized using ultravioletlight.DISTRIBUTION OF SDSPATHOGENS IN THE U.S.One of our objectives was to determinethe geographic distribution of SDSpathogens. The fungus o. narmari hadpreviously been reported to be the primarycause of SDS in Australia, but ithad not been found in North America.Ophiosphaerella korrae also was reportedto be widespread in Australia and wasdocumented as the cause of SDS inMaryland and California, whereas O.herpotricha was recovered from SDSpatches in Kansas and Oklahoma.However, these early studies providedan incomplete picture of the geographicdistribution of SDS pathogens in theUnited States because they were basedon a limited number of samples collectedfrom just a few geographic locations.Bermudagrass selections are screened for spring dead spot resistance in replicated field trialsin Stillwater, Oklahoma. Each plot was inoculated with Ophiosphaerella herpotricha, O. korrae,and 0. narmari. Several selections showed good resistance to the disease.Since 1994 we have intensivelysampled golf course fairways in severalstates and also collecteda small numberof isolates from widely dispersed geographiclocations throughout much ofthe range of where SDS occurs in theUnited States. Our survey indicates thatthere are regional differences in thedistribution of SDS pathogens. Themajority of isolates we collected inKansas and Oklahoma were o. herpotricha.Thisconfirms that this fungus isthe primary cause of SDS in the southernGreat Plains. The fungus O. korraewas isolated less frequently in thisregion, with almost all of these isolatescollected from a single golf course fairwaynear Afton, Oklahoma. Althoughonly a few o. narmari isolates wererecovered, they represented the firstreport of this fungus in North America.Ophiosphaerella narmari has since beenfound in other regions of the UnitedStates.16 GREEN SECTION RECORD

In contrast,O. korrae was the onlySDS pathogen in samples collected atsites in Mississippi,Alabama, SouthCarolina, Tennessee, and Virginia, and itwas dominant in North Carolina.Although the number oflocationssampled in each state was small, theseresults suggest that O. korrae is the mostwidely distributed SDS pathogen in thesouthern United States. Both O. herpotrichaand O. korrae were collected inKentucky, with their isolation frequencydependent on the location that wassampled.The distribution of SDS pathogensin the western United States is lessclear, although both O. korrae and O.narmari have been isolated from samplescollected near Los Angeles, California.Further sampling in Arizona, Nevada,California, New Mexico, and westernTexas is needed.The uneven distribution of SDSpathogens in the United States mayreflect regional differences in nativeranges of these fungi. While O. herpotrichahas been isolated from buffalograsslawns exhibiting SDS symptoms, it hasnot been isolated from this or othernative grasses in natural prairie stands.Therefore, we are still uncertain whetherthis fungus is native to the Great Plains.It seems unlikely that O. korrae isnative to North America since bermudagrassand Kentucky bluegrass (thefungus causes necrotic ringspot on thishost), the primary hosts for this fungus,are exotic grasses. An alternative explanationis that O. korrae was introducedand dispersed on infected bermudagrassroots and stolons. Thus, O. korrae mighthave initially infected an improvedbermudagrass cultivar adapted to aspecific geographic region and then wasdispersed via contaminated sodl sprigsacross a wide geographic area.A study by Wetzel et al. supports thishypothesis. They found that O. korraeisolates collected from several southernstates were genetically similar based onDNA fingerprinting techniques. Theseisolates differed substantially from O.korrae isolates collected from Kentuckybluegrass and bermudagrass in morenorthern regions of the United States.These northern isolates also weregenetically similar. We believe that O.korrae may have been introduced intoNorth America, with one or a fewinitial introductions occurring onbermudagrass in the southern UnitedStates and another introduction inmore northern regions on Kentuckybluegrass. Further genetic studies areneeded to confirm this hypothesis.Nevertheless, these results are strongevidence that this pathogen has beenmoved from one location to anotheron infected stolons or roots.AGGRESSIVENESSOF SDS PATHOGENSWe were also interested to see if therewere differences in SDS severity followinginoculations with O. herpotricha) O.korrae, and O. narmari. A two-year-oldstand of Midlawn bermudagrass atthe Kansas State University John PairResearch Center, Wichita, Kansas, withno previous history of SDS, was inoculatedin September 1997 with the threeSDS pathogens. All 14 sites inoculatedwith O. herpotricha developed symptomsin 1999. The patches reappeared and hadexpanded in diameter in May 2000.In contrast, only 10 of 14 sitesinoculated with O. korrae developedsymptoms in 1999, and of those only 7appeared the following year. Patchdiameters associated with O. korrae weresignificantly smaller than those causedby O. herpotricha. Unfortunately, thisstudy did not include any O. korraeisolates from the southern region. Onlyone of the sites inoculated with O.narmari developed SDS, and the patchdiameter of this spot was small.These preliminary results indicatethere are differences in aggressivenessof SDS pathogensTable IIdentification of Ophiosphaerella species isolated frombermudagrass cores collected in various states.to the bermudagrassNumber of IsolatesCollection O. O. O.Location Date herpotricha korrae narmariAlabama,Vestavia Hills 1999 0 20 0Arkansas, sites unknown 1994 0 6 0California, Los Angeles 1983,1999 0 2 4Georgia, sites unknown 1994-1996 0 3 0Kansas, 20 sites 1984-2000 55 0 IKansas, Independence 1994 71 9 0Kentucky, Mayfield 1998 2 13, 0Kentucky, Henderson 1998 14 5 IKentucky, Paducah 1998 0 17 0Mississippi, Starkville 1999 0 18 2Missouri, Dunklin County 1996 2 0 0North Carolina, 12 sites 1999-2000 0 63 INorth Carolina, Raleigh 1999-2000 12 5 0Oklahoma, Afton 1994-1996 201 38 22Oklahoma, Jenks 1994 173 0 0South Carolina, 8 sites 1999-2000 0 16 0Tennessee, Knoxville 1999 0 15 0Texas, Dallas 1996 3 0 0Virginia,Virginia Beach 1999 0 20 0Virginia, Charlottesville 1999 0 5 0Virginia, site unknown 1999 0 4 0West Virginia 1999-2000 00"MARCH-APRIL 2004 17

II' If c~, Table 2Development of spring dead spot on Midlawn bermudagrass following inoculationwith Ophiosphaerella herpotricha, O.korrae, and 0. narmari inWichita, Kansas.~1999 2000Number ofNumber ofinoculation Patch inoculation Patchsites with area sites with areaI:.' Fungal Species dead spots X (cm 1 )Y dead spots (cm 1 )Oph;osphaerella herpotr;cha 14 374a 14 1120aOphiosphaerella korrae 10 78b 7 264bOph;osphaerella narmar; I 46c I 79cSterile oats 0 0XNumber of 14 inoculation sites for each species in which spring dead spot symptoms developed.Plots were inoculated in September 1997 with three isolates of each species and were rated inMay of 1999 and 2000.YAverage patch area for those inoculation sites in which spring dead spot symptoms developed.Patch diameters not followed by the same letter are significandy different (P