nEWSLETTER OF THE TREE PRTHOLOGY COOPERRTIVE PROGRRm - U.O.F.5.

NO 10 NOVEMBER 1994

Tree Pathology Cooperative ProgrammeFor attention: Prof M. J. WingfieldDept. of Microbiology & BiochemistryUniversity of the Orange Free StateP.O. Box 339Bloemfontein 9300

Tel: 051 - 4012581Fax: 051 - 482004E-mail: mike@wwg3.univ.ac.za

In order for us to coordinate our services to you pleasehelp us by using the abovementioned contact address

Typical malformation of young Eucalyptus

grandis stems due to canker formation by

Botryosphaeria do thidea , following hail

damage.

DIRECTORS MESSAGE - THE YEARDRA WS TO AN ENDIn terms of diseases, this last year has continued to challenge usgreatly. In most areas the drought has continued to be severeand the pundits are predicting that we might expect the samefor the foreseeable future. Depressing news indeed. Of coursethis has a, significant effect on the opportunistic diseases that wehave to deal with. We are also finding that drought and otherstresses result in symptoms that we have to deal with. 'Ne arealso finding that drought and other stresses result in symptomsof some disease being more dramatic. In this case, I referspecifically to those pathogens such as Coniothyrium that appearto appear to infect trees irrespective of drought.

Although some of you mightfind it rather tiresome, ourmessage in every newsletterrequesting that samples or en-quiries be directed to a singlecon tact point, remainsoblivious to some. Let meexplain this need for this re-quest. Housing of the TreePathology CooperativeProgramme (TPCP) in aUniversity Department meansthat requests can go astray.\Ve believe that it is desirablefor members to be assured ofan response even when one ormore of us are "in the forests"or otherwise occupied by lec-tures and other duties. Wehave thus arranged that onephone number will always beassured of a reasonably rapidresponse. We also try to en-sure that every sample sub-mitted for diagnosis is loggedinto a database ensuring thatwe can track down thesesamples in the event of an en-quiry. \Ve :would thus bemost obliged if you 'wouldspread the word concerningthe official contact address ofthe group. In the end, this

will ensure that we are able toserve you more efficiently.

As many of you are aware,both Brenda and I have beenon sabbatical leave during thecourse of the past eightmonths. It would be whollyimpossible to summarise ourscientific experiences in thisnewsletter without making itlong and cumbersome to read.Suffice to say that we reallyappreciate having had theopportunity to expand ourknowledge and to learn newtechniques. In this regard weowe thanks to many peopleand organisations. Firstly,the University of the OrangeFree State for granting us sixmon ths of study leave andsome financial support: theFoundation for ResearchDevelopment who awardedboth of us study bursaries,Mondi Paper Co . whoawarded me a study bursaryand the Ernest OppenheimerTrust who also awarded me abursary. In addition to theabove, many of you had todeal with us via fax , e-mail or

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even snail mail. This was attimes inconvenient and wesincerely appreciate yourpatience!! Finally many TPCPteam members including stu-dents and particularly GertKemp had to assume extraduties. These were executedmost competently and wereally appreciate this supportvery much.

While on the subject of sab-baticalleave, I might mentionthat Brenda has returned withsome wonderful techniques toidentify specie of the impor-tant tree pathogen Armillaria .This is going to provide uswith enhanced insight into thefungus and its biology inSouth Africa in the near fu-ture. \Ve will be communi-cation more on this subjectboth in this newsletter and inthe future.

In this newsletter we will treata number of topics that havebeen raised by many of youduring the course of thisyear. Perhaps one of themost important of these is"What do we really knowabout Coni ot hyr iurn'!"Another topic of current andintense interest concerns thebiology of Botryosphaeria andSphaerop sis . In both thesecases we have accumulatedconvincing evidence that thesepathogens are able to live in alatent (non-infective) phase inhealthy trees. This of coursehas a very substantial impact

on how we view the associateddiseases and on how theymight be managed. Anothertopic of current concern re-lates to our projects and ac-tivities as a whole. Becauseof the severe impact of someof certain eucalypt diseasesespecially in Zululand , thereis an unfortunate misconcep-tion that most of our energyand time is spent in that area.Nothing could be furtherfrom the truth. Indeed it

would be fair to say that ourtime and activity is quitebroadly spread across theforestry areas of South Africaand there remains a very sub-stantial input into pine dis-eases. In order to assurethose of you working in areasother than in Zululand , I amincluding a break-down of ourprojects and activities in thisnewsletter.

And finally - this is the

second and last newsletter ofthe year. It must thereforeserve to convey our thanks toall of you that have assisted usduring the course of the yearwith collection of data, set-ting up of field trials and inmany other ways. We appre-ciate this assistance most sin-cerely. \Ve also all join inwishing you a very happy andjoyous festive season and agreat 1995.

TISSUE CULTURE: A TOOL FOR SCREENINGPLANTS FOR DISEASE RESISTANCE

'When sections of a plant suchas pieces of leaf or rootmaterial, shoot nodes orembryos are placed onnutrient media, containingplant hormones, callus isformed. Callus is a mass ofundifferentiated, dividingcells, and can be subculturedon to fresh media producinggenetically-identical material.By altering the plant hor-mones and their concentrationin the medium, root andshoot induction will takeplace. The procedure takes arelatively short space of timewhen compared to "normal"propagating methods. Theideal stage to screen for dis-ease resistance is when the

callus has formed. A numberof methods can be used andtwo will be described here.

The easiest method is to in-oculate the callus with fungalspores. One would expectthat in the susceptible interac-tion, colonization of the cal-lus will take place whichwould not occur in the resis-tan tltoleran t interaction.The success, however, willdepend on the particular fun-gus/host examined.

Another method would be toeither expose the callus cellsto a fungal toxin directly (in-oculate callus with the toxin)or indirectly (grow the callus

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on media containing thetoxin).

One of the essential factorswith screening plants for dis-ease resistance using tissueculture is to correlate resultswith field studies. For ex-ample, a tree with thick barkwill have a natural defencemechanism not expressed intissue culture.

The TPCP is currently con-sidering making use of tissueculture. It would be used as ascreening technique for arange of plant pathogens. Inthis way, resistant or tolerantmaterial would be rapidlyidentified .

POPULATION DIVERSITY AMONG BRAZILIANISOLATES OF CRYPHONECTRIA CUBENSIS

Cryphonectria cubensis is oneof a notorious group of cankerpathogens of Eucalyptus treesand causes serious stemcankers in Brazil and manyother tropical areas of theworld. This pathogen haslimited the development ofplantations of susceptibleEucalyptus spp. in areaswhere climatic conditionsfavour disease development.The disease is favoured byhigh rainfall (2000-2400mm/yr .) and temperaturesabove 23 C. In the cooler,drier areas of Brazil infectionrates are much lower as is the

extend of canker develop-ment.

Results of a recent TPCPstudy on C. cubensis sug-gested that isolates of C.cubensis in South Africarepresent a uniform popula-tion structure. This is indica-tive of an introducedpathogen. Therefore, theaim of this study was to deter-mine the genetic variabilityamongst Brazilian isolates ofthe pathogen. Isolates of C.cubensis were collectedthroughout the Eucalyptusgrowing areas of Brazil and

were compared based on theirvegetative compatibility reac-tions. Based on the number ofvegetative compatibilitygroups (VCG's) found in thisstudy, there appears to be ahigh degree of diversity in theBrazilian population of C.cubensis . This was not unex-pected given the abundantpresence of the teleomorph inBrazil as opposed to in SouthAfrica. These results suggestthat C. cubensis has beenpresent in Brazil for an ex-tended period of time and thatthe fungus has only recentlybeen introduced into SouthAfrica.

Fig 1: At the end of the incubation time, mycelia that werevegeta~ively compatible had grown together, forming aconfluent mycelium. Incompatible mycelia had grown to ameeting point in the agar, but remained separated by a"barrage-like" reaction line formed along the line of contactbetween paired colonies.

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SIREX WOOD WASP - THE FUNGAL COMPONENTAs many of you are now aware, the potentially very seriouswood wasp Sirex noctilio has recently been discovered in SouthAfrica by Geoff Tribe and his staff at the Plant ProtectionResearch Institute, Rosebank , Cape. This insect pest hascaused tremendous damage to plantations of Pinus radiaia inAustralia and New Zealand in the past and is currently active inArgentina and Brazil. We have predicted and feared its intro-duction into South Africa for many years and its appearance hernow is most discomforting.

One of the most fascinatingaspects of this pest, at leastfrom the pathologists view-point, is the fact that it livesin a strict symbiosis with afungus. Indeed it is the fun-gus and a mucous depositedby the wasp that is believed toultimately kill the trees al-though this is perhaps a mootgiven the fact that the two arefully dependent on eachother. The fungus, Amy/os-tereum areol atum is aBasidiomycete. This meansthat it is related to the mush-rooms as opposed to, for ex-ample Cryphonectria which isin the distantly related As-comycete class.

Amylostereum areolatum istransmitted from tree to treeby female Sirex wasps. Thisfungus is carried in specialised"transport sacs" known asmycangia which are associatedwith the ovipositor. Thuswhen the insect lays her eggsby means of boring into thebark of trees, the fungus is

also transmitted. The fungusthen has an association with amucous substance that isdeposited with the eggs andthen grown into the wood,blocking the vessels andresulting in wilting of in-fected trees.

A very successful strategy formanaging Sirex infestations isthrough biological control ofthe insect with a parasiticnematode. This nematodewhich has been used very suc-cessfully in Australia andNew Zealand is known asDeladenus siridicola . Femalewasps infested with thenematode lay eggs that carrythe namatodes which are thentransmitted to trees. Thesenematodes then feed on thefungus Amylo st ereumareol atum and multiplyrapidly in trees. Later an in-fective stage of the nematodeinfects wasp larvae which arein effect rendered sterile.Once again, the fungus has avery important role to play in

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the biological control of thewasp.

A fascinating aspect of thewood wasp in areas where ithas been introduced as an ex-otic relates to the geneticdiversity of the wasp. Logi-cally, a more diverse popula-tion in the wasp will be lessintensively affected by thenematode in prospectivebiological con trolprogrammes. One rather fas-cinating approach to measur-ing the diversity of the wasppopulation will be through astudy of the fungus which itcarries. Genetic diversity offungal populations are rela-tively easily studied and thetechnology to deal with suchquestions has been developedby researchers in the TPCP inrecent years. We thereforehope to capitalise on this ex-perience and, in collaborationwith Geoff Tribe of thePPRI, consider genetic diver-sity (as well as other aspects)of Sir ex noct ilio in SouthAfrica.

THE 1994 AMERICAN PHYTOPATHOLOGI-CAL SOCIETY'S ANNUAL MEETING -TPCP

INVOLVEMENTThe annual meeting of theAmerican PhytopathologicalSociety (APS) is the largestannual gathering of plantpathologists in the world withapproximately 2000 plantscientists from all over theworld attending annually.The cultural mix of attendeeswith diverse backgrounds andvarious interests makes themeeting unique. This year'smeeting took place in Albu-querque , New Mexico'slargest city. from 6-10August. The theme ;for themeeting was 2)Pro PlantPathology" . The word "pro"reinforced the positive. profi-cient. professional attitude ofthe plant scientists whogathered at Albuquerque.

The TPCP was represented atthe 1994 APS annual meetingby mr Gert Kemp and drWijnand Swart . The meetingwas preceded by a one-dayforest pathology tour or-ganised by the Forest Pathol-ogy Committee of the APS.The tour travelled north ofAlbuquerque through pueblocountry through the Jemez

Mountains to Bandelier Na-tional Park. Pathology topicsmainly focused on ripariantree health, dwarf mistletoemanagemen t , hazard treemanagement and stumpremoval trials for Armillariaroot diseasecontrol.

Topics in the technicalprogramme of the APS meet-ing reflected many new andexciting developments in thescience of plant pathology.The Forest Pathology com-ponent of the meeting in-cluded two poster sessionscomprising 17 posters in totaland two oral sessions with 23papers in total. The TPCPmade four contributions tothis programme. A paper byA. J. Cilliers» W. J. Swartand M. J. Wingfield entitled,"Infection of P. elliottii seedby L. theobromae provided forsome very stimulating discus-sion. Poster contributionswere by: I. P. van der Wes-th uizen , M. J. Wingfield •W . 1. Swart and G. H. J .Kemp entitled, 2)Effect ofwater stress on pathogenicityof twoEucalyptus canker

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pathogens"; I.P. van derWesthuizen. W. A. Smit ,M.J. Wingfield and G.H.J.Kemp entitled."Hypovirulence associatedwith dsRN A discovered inCryphonectria cubensis"; andW. J. Swart , W. C. Saaimanand W-M. Botes entitled."Use of the isolated root capcells and cell cultures fromPinus spp. in an assay fortoxin production by Sphaerop-sis sap inea . Two very en-lightening discussion sessionswere also included in theForest Pathology programmenamely, "The application ofGIS/GPS technologies toForest Pest Management" andMolecular Techniques inForest Pathology2)1 The an-nual APS meeting alsoprovided an opportunity forthe annual meeting of theAPS Forest Pathology Com-mittee. Both Gert and Wij-nand» who is a member ofthis committee for the nextthree years. attended thismeeting which provided aninteresting insight to the ac-tivities and concerns of forestpathologists in the USA.

BLACK BUTT OF'ACACL4 ilfEARNSHBlack butt disease is eh a-r act er ised by black discol-oration of the bark at the baseof black wattle trees. Thissymptom is accompanied bythe cracking of the bard andthe exuding of gum (Fig. 1).The di se ase occurs in allwattle growing areas in thecoun try. The causative or-ganism was identified asPhytophthora parasitica (Das-tur .) Water house in the past,although It was generallyrecognised that this is a more

complex problem, In manycases the black discolorationsspreads higher up the stem ofinfected trees towards the ter-minal growth points (Fig. 2).Isolations from these advancedlesions have failed to yield P.parasitica . Various secondarypathogens have however beenisolated in our preliminarysurveys. At this stage webelieve that P. par asitica In-fects trees through the rootsor base of the tree, causin gentry sites for opportunistic

pathogens to infect. Secon-dary lesions then spreadhigher up the tree. Atpresent we are conducting in-oculation to determine the se-q uence of events associatedwith the development of blackbutt disease. This will in-cl ude inoculations withvarious fungi that have beenisolated. We hope then toalso develop strategies toselect trees that are able totolerate the problem.

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SEIEC1ED ABS1RACTS FROM PRESENFAlIONSBY MEMBERS OF THE 1PCPAT RECENF CONFERENCES

BOTRYOSPHAERIA D011IIDEA ENDOPHYTIC ON EUCAIYP-TU S N ITEN SAND EUCALYPIUS GRANDIS IN SOUlH

AFRICA

Species of Botryosphaeria are well recognised as opportunistic, wound related pathogenswith a wide host range. In South Africa, B. dothidea is an important pathogen ofEucalyptus, causing serious die-back and canker diseases. These diseases have been as-sociated with trees suffering from severe environmental stress, such as drought. AlthoughB. dothidea is a wound related pathogen, drought stress usually does not result in visualwounds. The aim of this study was to determine whether B. dothidea might be en-dophytic, in the leaves and xylem of healthy E. nitens andE. grandis trees, only becom-ing active at times of stress. Fungal isolates representing many genera were isolated fromleaf and xylem segments, with B. dothidea being the only dominant species known to be aserious pathogen. Results clearly indicate that B. dothidea is an endophyte of E. nitensand E. grandis and its association with disease development in drought-stressed trees re-quires further investigation.

PY1HIUM IRREGUURE ASSOCIATED WITH HNUSSEEDLING DEATH ON PREVIOUSLY CULTIVATED

LANDS

A serious root disease of Pinus patula seedlings has occurred in the northeastern Capeforestry region of South Africa during the past 3 years. Mortality as high as 100% was ex-perienced 4-5 months after seedlings were planted on previously cultivated agriculturallands. No mortality, however, occurred in plantings on virgin lands. Where seedlingssurvived on previously cultivated lands their growth compared poorly with those on virginlands. Pythium irregulare was consistently isolated from diseased roots of P. paiula as wellas from soil of previously cultivated lands. P. irregulare was highly virulent when artifi-cially inoculated onto 4-mo-old P. patula and 2-mo-oldEucalyptus grandis seedlings. P.irreguiare , therefore, appears to be an important factor associated with deaths of P. paiulaon previously cultivated agricultural lands .

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CHARACTERIZATION OF AFUSARIUMSP. FROMGLADIOLUS PATHOGENIC TO PINES

Fusarium subglutinans is a well-known pathogen of many crop plants. The fungus alsoca~ses a serious disease of pines known as pitch canker. It has been proposed that isolatesfrom pines be designated F.subglutinans f. sp. pini (FSP) . An enigma regarding this fun-gus is that some FSP isolates have been reported to induce decay of gladiolus corms. Iso-lates of F. subglutinans from gladiolus were found to be weak to moderately pathogenic topine seedlings. In this study we re-examined previously studied isolates from gladiolus inthe U.S.A. that had been treated asF. subglutinans in the literature. We found thatthese isolates were typical of F. proliferaium, They were mildly pathogenic on pine see-dlings and significantly less virulent than an isolate of FSP. Inoculation of gladiolus cormswith these isolates of F. proliferaium from gladiolus, F. oxysporum from pine seedlingsandF. subglutinans from pines (PSP), mango, maize and pineapple indicated that onlyisolates of F. proliferatum were moderately pathogenic to gladiolus corms. This studytherefore confirms that isolates of F. subglutinans from pines represent a specific formaspecialis within the species.

EFFECf OF WATER STRESS ON PATHOGENICITY OFTWO EUCAlYPTUS CANKER PATHOGENS

Eucalyptus species are extensively planted by the South African forestry industry.Cryphonectria cubensis and Endothia gyrosa are two closely related canker pathogens ofEucalyptus. Cryphonectria cubensis is restricted to areas of high rainfall and temperaturewhereas E. gyrosa has a wider distribution. The aim of this study, was to determinewhether a relationship exists between virulence of these fungi and water stress. Artificialinoculation trials were conducted with both pathogens on trees under stress as well as underconditions of normal water availability. Stress conditions were regulated by using a pres-sure bomb. Trees were more susceptible to C. cubensis under non-stressed conditionswhile the opposite was true for E. gyrosa, This is consistent with field observations whereC. cubensis is more severe in areas of higher rainfall and E. gyrosa under conditions ofdrought stress.

IMC5

The Fifth InternationalMycological Congress washeld in Vancouver,Canada from the 14th untilthe 21st of August 1994 atthe campus of theUniversity of BritishColumbia. Among theattendants were thefollowing from the UOFS:

Prof M.l Wingfield, Dr.B.D. Wingfield, Mr. GertKemp, Adriaan Smit(Infruitech), ChristaVisser, ChristopherViljoen and CorliStrydom. Papers werepresented by the aboveattendants as well asvarious students (CelesteLinde, Altus Viljoen andHenk Smith) in absentia.

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As one of the delegates Ifound the congress anincredible learningexperience. It was alsovery rewarding to learnfrom internationallyknown researchers thatour work is on a par withtheirs even though we aresomewhat isolated.

HYPOVIRULENCE ASSOCIATED WITH dsRNADIS-COVERED IN CRYPHOlvECTRIA CUBENSIS

Cryphonectria cubensis is responsible for serious losses in commercial Eucalyptus planta-tions around the world. Currently) the only means of controlling the disease is by resis-tance breeding and selection. The virulence of certain fungal pathogens can be reducedbiologically through hypoviru lence , as is true in the closely related pathogen C.parasitica . Isolates of C. cubensis were screened for the presence of dsRNA and otherhypovirulence associated traits. Pathogenicity tests were also conducted to link thepresence of dsRNA with hypovirulence. Various C. cubensis isolates from South Africawere found to contain dsRNA.. These were hypovirulent in comparative pathogenicitytests and also displayed other hypovirulence associated traits such as a reduction in oxalicacid production and laccase activity. Results further indicate that naturally occurringhypovirulence might play an important role in reducing the impact of Cryphonectriacanker in South Africa.

PUBLICATIONSLinde, C. '. G .H. J. Ke~p and ~-1.J. Wingfield. 1994. Diseases of pines andeucalyptu.s In South Africa associated with Pythium and Phytophthora species.South African Forestry Journal 169:25-32.

Viljo~n, A. , :tvI: 1. Wingfield and "\V.F. O. Marasas , 1994. First report ofFusarium subglutinans f. sp. pini on pine seedlings in South Africa Plant Disease78: 309-312. .

Li~de) C ..' G ..H. J. Ke~p and 1-1.1. "\Vingfield. 1994. Pythium irregulare as-sociated WIthPinus seedling death on previously cultivated lands Plant Disease 78.1002-1005. . ..

Linde~ C. , C? .H.J ..Kemp an.d1-1.J. Wingfield , 1994. Root disease of Eucalyptusgrandis associated WItha species of Pythium. Plant Disease78:1006-1009. .

'Yingfie~d). :tvI.J. and W. J. Swart. 1994. Integrated management of forest treediseases In South Africa. Forest Ecology and Management 65: 11-16.

Lin~, C. , <? H. J ..Kemp and 1-1.J. Wingfield. 1994. Pythium and Phytophthoraspecies associated WItheucalypts and pines in SOuth Africa. European Journal ofForest Pathology 24:xxx-xxx(ln press).

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