P A L M S Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

ObservationsandPathogenicityExperimentson Gsnodermcrzonatum inFloridaGanoderma butt rot of palms, caused by Ganoderma zonatum, is awidespread and serious disease in Florida, USA. The means by which thefungus invades healthy palm trunks is not known. However, infectionappears to begin at the center of the base of palm trunks and spreadsoutrvard and upwards from there, eventually producing a hard, shelf-likestructure that is the basidiocarp (conk). Basidiospores produced withinthe conk may be the primary method for spreading the fungus in theurban landscape. Wilt symptoms are caused by the fungus rotting thexylem, as well as other trunk tissue, in the lowest 1.5 m of trunk. It isbelieved that most, if not all, woody palms are susceptible to this disease.Experimental attempts to inoculate healthy trees with this fungus havegenerally been unsuccessful.

Palms are found throughout Florida (USA) in bothnatural and landscaped settings. They are not lgrown for agronomic purposes, but are importantas ornamental plants. When a list of the top ten ,diseases of Florida ornamentals was compiled in ,1997, Ganoderma butt rot of palms was listed as ,number two (Schubert et al. 1997). The fungal ,genus Ganoderma is a group of wood-decaying ,

M. t. EluorrAND

T. K. BnoscHer

University of Florida

Fort Lauderdale Research andEducation Center

3205 College Avenue

F ort Lauderdale, Floricla

33314 USA

basidiomycete fungi that are found throughoutthe world on all types of wood - gymnosperms,hard- and softwood dicots, and palms (Gilbertson& Ryvarden 1986). The genus is greatly debatedat the species level; Miller et al. (1999) describedit as "currently chaotic." This has been due tospecies names being added and then dropped, aninconsistency in criteria used for identification

62 PALMS 45(2): 62-72

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1 (above). Sabal palmetto (sabal palm) with wil ted anddesiccated leaves due to Canodermo zonotum infection.

2 (r ight). Syogrus romonzoff iana (queen palm) dying fromCanoderma zonatum. Only the spear and one other leafremain green.

3. Basidiocarp (conk) o1Conoderma zonatum.Note g lazed redd ish-brown top surface andwhite undersurface. The"s t ra igh t " s ide o f theconk is direct ly attachedto the t runk . There is no"s tem" or "s ta lk " tha tattaches the conk to thetru n k.

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PALMS Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

purposes at the species level, and the fact thatmorphological characteristics change based onenvironment (Moncalvo et al. 1995).

In Florida, Ganoderma butt rot is a lethal diseasethat appears to be caused by the fungus Gqnodermazonatum Murrill. Another fungal name that wasassociated with this disease in the first half of the20th century was Ganoderma sulcatum MurrilI(Murrill 1902,79O8; Childs & West 1953). Murrillindicated in his papers that both species wereassociated with dead wood in Florida, and thatthey were very similar in appearance. Recently,these two species have been grouped together asone, G. zonatum (Gilbertson & Ryvarden 1986).Although G. zonatum is often referred to as amember of the G. lucidum complex, it is a distinctspecies from G. lucidum (Adaskaveg & Gilbertson1989). Information is available regarding themorphological and cultural characteristics of G.zonatum (e.g., Adaskaveg and Gilbertson 1989;Adaskaveg and Gilbertson 1988; Gilbertson &R1'varden 1986), but little is known about thedisease it causes. A similar basal stem rot of oilpalms is widespread, but is caused by a differentGanoderma species, presumed to be G. boninense(Miller et al. 1999). This pathogen does not occurin North America. Considerable information isavailable on this disease in Southeast Asia, so wehave relied heavily on this information as a basisfor initiating research in Florida. This article is acompilation of our personal observations andpathogenicity research results obtained since 1994.

I. OBSERVATIONS

Symptoms and Signs

Ganoderma zonatum is a white rot fungus thatproduces numerous enzymes that allow it todegrade (rot) woody tissue, primarily lignin andcellulose (Adaskaveg et al. 1991). As the fungusdestroys the palm wood internally, the xylem(water-conducting tissue) will eventually beaffected. Therefore, the primary symptom thatmay be observed is a wilting, mild to severe, of allleaves but the spear leaf (Figs. 1 and 2). Othersymptoms can best be described as a generaldecline - slower growth and off-color foliage.However, these symptoms alone should not beused for diagnosis of Ganoderma butt rot, sinceother disorders or diseases may also cause thesesymptoms. Only when these symptoms areaccompanied by the development of thebasidiocarp (conk) (Fig. 3) can the palm beconfirmed as affected by G. zonaturu. Also, it iscommonly observed that basidiocarps will formprior to any obvious wilting or decline symptoms.

It should be emphasized that this fungus is notrestricted to vascular tissue. The palm wilts because

the vascular tissue as well as the surrounding tissuehas been degraded by the fungus. This becomesevident when a palm that has died or is dyingfrom Ganoderma butt rot is cut down and cross-sections made of the trunk. Figs 4 and 5 areexamples of the wood rotting and diseaseprogression pattern observed. The funguscolonizes and degrades the palm trunk tissueclosest to the soil line first, expands in diameterat the base and moves up the center or near-centerof the trunk. Therefore, the disease progressionpattern within the trunk is best described as cone-shaped, widest at the soil line and narrowing to apinpoint. In general, we do not see the fungusextend more than 1.5 m (-5 ft) up into the palmtrunk. Although roots may eventually rot,especially after the palm dies, the lethal damagefrom the fungus appears to be associated with thetrunk degradation. Ganoderma butt rot is a diseaseof mature palms (i.e., palms with trunks) and hasnot been observed to affect seedling or juvenilepalms in natural or landscape settings.

It was previously thought that the location of theinitial basidiocarp formation was the entry pointfor the fungus, perhaps from a wound to the trunk.We do not believe that is true. The location of thebasidiocarp is where the fungus is emerging fromthe trunk. This means the degradation is occurringinternally as the fungus moves from the lowercenter of the palm to the outside. This is especiallyevident when a palm suspected of being infectedwith G. zonatum, based on wilt symptoms andproximity to other diseased palms, but withoutbasidiocarps is examined. This means that woundsare not a likely factor in disease initiation. Otherexternal environmental factors associated with thetrunk are probably not associated with diseasedevelopment either, such as too much mulcharound the trunk, irrigation heads striking thetrunk, flowers or shrubs too close to the trunk, orpainting the trunk.

Life Cycle of the Fungus

The basidiocarp or conk is the most easilyidentifiable structure associated with the fungus.The conk is a specialized mass of fungal growththat originates from mycelia in the palm trunk.Fig. 6 i l lustrates different stages in thedevelopment of the basidiocarp. When the fungusfirst starts to grow on the side of a palm trunk orpalm stump, it is a solid white mass that isrelatively soft when touched. It will have anirregular to circular shape and is relatively flat onthe trunk or stump.

As the conk matures, a small shelf or bracket willstart to form as the basidiocarp begins to protrudefrom the trunk. It will still be white, both on the

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P A L M S Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

Thble 1. Palms documented to have been infected with Ganodermq zonqtum.

Palm

Acoelorraphe wrightiiAcrocomia aculeataAdonidia merrilliiAiphanes sp.Arenga engleriArenga tremulaArenga undulatifoliaAttalea sp.Bactris maiorBrahea berlandieriBrqhea brandegeeiBrqhea dulcisBrahea edulis x brandegeeiButia eriospathaButia capitataCarpentaria acuminataCaryota mitisChamaerops humilisCoccothrinax sp.Cocos nuciferaCopemicia curtisiiDictyosperma albumDypsis cabqdaeDypsis lutescensElaeis guineensisEuterpe edulisGastrococos crispaHyophorbe indicaLivistona benthamiiLivistona chinensisLivistona merrilliiLivistona muelleriLivistona saribus

Sourcez

FTGFTGFTGDPIFTGFTGFTGFTGFTGFTGFTGFTGFTGFTGDPIFLRECDPIFTGFTGDPIFTGDPIFTGDPI, FTGTRECDPIFTGTRECFTGDPI, FTGFTGFTGForestry

FTGDPI, FTGFLRECDPI, FTGFTGFLRECTRECDPI, FTGFTGFTGFTGDPI, FTGFTG, TRECFTG

Nannorrhops ritchiqnaPhoenix canartensisPhoenix dactyliferaPhoenix reclinataPhoenix roebeleniiPhoenix sylvestrisPtychosperma elegansPty cho s p erma m ac arthuriiPty cho sp erma s qlomonens eRoystonea altissimaRoystonea oleraceaRoystonea regiaSabal causiarumSabal mauritiiformisSabal palmettoSabal uresanaS atqkentia liukiuensisSerenoa repensSyagrus oleraceaSyagrus picrophyllaSyagrus romanzoffianaSyagus sanconaSyagrus schizophyllaSyagrus x costaeWashingtonia robusta

DPI, FLREC,FTGFLRECFTGFTGFTG

DPI, FLREC,FTGFTGFTGDPI, FTG

ZDPI = Diseases and Disorders of Ptants in Florida(Alfieri et aI., 1994); FTG = Fairchild TropicalGarden, Miami, FL; TREC = University ofFlorida's Tropical Research and EducationCenter, Homestead, FL; FLREC = University ofFlorida's Fort Lauderdale Research andEducation Centeq Fort Lauderdale, FL; Forestry= Florida's Department of Forestry, Miami, FL.

top and bottom surfaces. Eventually, it will forma very distinct shelf-like structure that is quitehard with aglazed reddish-brown top surface anda white undersurface (FiS. 3). A mature conk willhave distinct zones, hence the name G. zonatum.The basidiocarp will have a half-moon shape withthe "straight" side directly attached to the trunk.This is referred to as a sessile basidiocarp asopposed to one that has a stalk or stipe (Figs 3 and6). Conks can be up to 20 cm (8 in) at their widestpoint and 5 cm (2 in) thick. Microscopicbasidiospores wil l be produced in the "pores"

present on the underside of the conk. Whenbasidiospores are dropped en mass on a whitesurface, they will appear brownish-red in color.

The conk is considered an annual conk. Once itstops growing, it does not begin growing and

expanding in size at a later date. In southern, sub-tropical Florida, conks can be producedthroughout the year. In northern, temperateFlorida, conks are normally only produced duringthe warmer months of May through October. Wehave observed thdt:"mature, viable basidiosporesare produced very eitrly in the formation of thebasidiocarp. Spores were produced shortly afterthe conk began to protrude (form a shelf from thetrunk, while it was still white, and long before thetop turned a reddish-brown color. We believe thatthe spores are the primary means of spread of thefungus, but that has yet to be proven. Recentresearch with Ganoderma in Malaysia determinedthat G. boninense may spread in oil palm plantingsthrough basidiospore dispersal or through contactwith long-term residual inoculum (mycelia, etc.)in soil debris (Miller et al. 1999). The genetic

6 5

l ) , \ 1 . \ l s C a n o d e r m a i n F l o r i d a : E l l i o t t & B r o s c h a t V o l u m e 4 5 ( 2 ) 2 0 0 1

I

I-

trL:,.. \.

11.. . : , , , . - ' - -*+

Fig. 4 ( top). Cross sect ions of lower l runl< of Syogrus rornctnzoI f iano intested wi th Coiror le i into zot lotut ] ) . Top- let t sect ion is

bo t t om sec t i on ( sec t i on 1 ) and rema in i ng sec t i ons p ro l 1 ress up t he l r unk . No te da rken inq o f wood due t o f unga deg rada t i on

( ro t ) . F i g . 5 ( bo t t o r r ) . Sec t i ons 5 and 6 o f F i g . ' 1 ( bo t t om row , r n i dd le ) a f t e r r nc r : ba t i on i n a mo i s t chamber t o r ' 1 days .

White growth is mycel ia of Conoderrno lonotL. t t l and corresponds wi th the discolored area of the cross-sect iot- ts .

:l .:

'^i'*

.+'{,' r \ ' '

*Tii

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P A L M S Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

. r'r't ,; f)'^t i 'rNl.^, ';r,f '*i \/t.r#'i"r

.i,; lrj

71;l i*fiFig.6 (top). Three phases of basidiocarp (conk) development of ianoderma zonotum. The white "button" near the topof picture is the beginning stage of the conk. The lower-r ight structure is a mature conk. The lower-left structure is alsoa mature conk, but i t is an old one; the underside of the conk is no longer white. Fig. 7 (bottom). Cut palm stump withnumerous basidiocaros of Gonodermo zonotum formino on it.

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information obtained by this research groupsuggests that the fungus does not spread by directroot-to-root contact between palms.

The diversity and isolation of locations in Floridawhere Ganoderma butt rot has occurred wouldsupport the concept of spores as a primary methodof spread. Palms are not a plantation crop inFlorida, but are found in two distinct settings. Onewould include natural groves of non-transplantedsabal palms (Sabal palmetto). The other is the urbanlandscape, where the majority of palms aretransplanted from either natural settings inFlorida, date groves from south-western USA, orplant nurseries. In urbanized southern Florida, the"soil" upon which developments are built, and inwhich palms are transplanted, is dredged fromman-made lakes. It is devoid of any possible plantdebris harboring G. zonatum. However, onlygenetic studies will definitively demonstrate howG. zonatum spreads.

After spores are released, it is unclear exactly whathappens next. We do not know if the sporesbecome dormant in nature. In a laboratory setting,the spores do not show any dormancy andgerminate readily on media after being droppedfrom the basidiocarp. However, in the real world,they may indeed become dormant or need specificsubstrates, temperature, relative humidity and soforth for germination to occur. We do know thatat some point in time multiple spores dogerminate, mate and produce mycelia that will gothrough three distinct stages of development -primary, secondary and tertiary. The basidiocarpor conk is composed of the tertiary mycelia.

Host and Geographic Range

In the Diseases and Disorders of Plants in Florida(Alfieri et al. 1994), five non-palm species are listedunder G. zonatum. It is quite possible that G.zonatum was not a pathogen of these non-palmspecies, but rather a saprobe that grew on thewood after the tree had died. According toAdaskaveg et al. (1991), the only authenticatedreports of G. zonatum are from palms. Howeveqthere has been one report in Florida ef G. zonatumassociated with a cycad. Still, palms should beconsidered the primary hosts of this fungus. Table1 lists all of the palms known to have died fromGanoderma butt rot in Florida. This l ist wascomoiled with information obtained from.tumerous sources in Florida, as indicated in thetable. Although not all palm species known to begrowing in Florida have succumbed to this disease,the majodty of those species not on this list arerelatively uncommon in Florida. Therefore, justbecause a palm is not listed in this table, it is notsafe to assume that the oalm is resistant to the

disease. In fact, it is probably more Iogical toassume that all palms are susceptible to G.zonatum. The only possible exceptions would bepalm species that do not form woody trunks -

e.g., Sabal minor, some Chamaedorea spp. Since G.zonatum kil ls by degrading wood, these palmspecies may not have any suitable tissue to serveas a substrate for the fungus.

The distribution of G. zonatum in Florida is shownin Fig. 8. This information was obtained frommany of the same sources as listed for the palmhost range and by contacting University of FloridaCooperative Extension Service faculty in eachcounty. While the disease is more prevalent in thesouthern half of the state where palms are ingreatest abundance, it is certainly not restricted tothat area.

Environments or Practices that EncourageDisease Development

To date, we have observed no common environ-mental conditions or landscape managementpractices that favor the development ofGanoderma butt rot. The disease has beenobserved in natural settings (palms nevertransplanted) and in highly-maintained, trans-planted landscapes. It has been observed on palmsthat have been maintained very well nutritionally(no nutrient deficiencies) and on palms that wereseverely stressed by deficiencies. The disease hasbeen observed in well-drained settings and inswamps. The fungus has killed trees that had noapparent mechanical injuries and those that hadbeen severely damaged by weed trimmers. Soilt)?e appears to have no relationship with diseaseeither as diseased palms have been observed ondeep sands (both silica and calcareous), muck(peat), and limestone rock. There has been nosimple discernible pattern to provide clues as towhy palms become infected and die from G.zonatum.

Management Considerations

In general, the fungus will be located in the lower1.5 m of trunk. This has two implications. First,this means that only'ihis lowei trunk portionshould not be chipped and used for mulch. Ifpossible, the diseased section should be placed ina legal landf i l l or inc inerated. The remain ingportion of the palm trunk could be immediatelychipped and used for mulch in the landscape.

Secondly, only the lower 1.5 m of trunk will needto be protected from the fungus. However, typicalxylem-limited, systemic fungicides will not beeffective unless they are capable of spreadingbeyond the vascular tissue and protecting all thewood in the lower portion of the trunk. No

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P A L M S Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

fungicide will be effective once the basidiocarpshave formed, since a large percentage of the trunkis already destroyed. Since we have no means ofpredicting or determining which palms areinfected with G. zonatum, this effectivelyeliminates the use of fungicides as a controlmethod, either preventively or curatively, for thepresent time.

Since basidiospores are probably the primarymethod of spreading the fungus, we urge peopleto monitor their palms closely, especially after apalm has died or been removed for anlz reason.The fungus will readily colonize and degrade palmstumps or dead palm trunks (Fig. 7). Once itbecomes established on this dead wood, it willnormally produce basidiocarps with millions ofbasidiospores. We had never observed G. zonatumas a pathogen on the Fort Lauderdale Researchand Education Center (FLREC) property unti lMarch 1993 when it first killed a Sabal palmetto.Interestingly, this palm was only - 30 cm awayfrom a sabal palm stump, left when a healthy palmhad been cut down 5 years earlier for researchpurposes. Howeveq we had observed G. zonatumbasidiocarps on this stump as early as August 1991.More recently at the FLREC, we observed G.zonatum basidiocarps on dead Carpentariaacuminata trunks. These palms had died fromother causes. We also noticed that there was aCarpentaria palm in this same planting, - 1 maway, that appeared wilted. When it was cut down,the center of the basal trunk area was alreadyrotted from G. zonatum. We have also observedand been told on numerous occasions ofGanoderma butt rot occurring on Dypsls lutescens(areca palm) after some of the mature canes hadbeen removed. In all probability, the fungus movesin on the dead stumps of these palms and spreadsfrom there to living canes.

Therefore, monitor your palms in Florida, andother areas of the south-eastern USA where thefungus has been documented, for the basidiocarps.Remove the basidiocarp (conk) and place in a trashreceptacle that will be incinerated or delivered toa landfil l . Do not place in trash that wil l berecycled in the landscape. The earlier thebasidiocarp is removed (i.e., before it becomes adistinct shelf-like structure), the less likely thatspores will be released into the environment. If;zou have never observed Ganoderma butt rot onthe property, monitoring the palms once everysix months will be adequate. Once you haveobserved the basidiocarps on palms or have a palmcut or fall down for any reason, monitor the palmsat least once a month.

It has been observed repeatedly that a replacementpalm planted into the same site where a palm died

from Ganoderma butt rot also eventuallysuccumbs to this disease. This would indicate thatthe fungus does survive in the soil, either asinfected plant material or spores. Therefore, it isnever recommended to plant another palm in thesame location as the previous Ganoderma-infestedpalm. In general, no other plant tree species grownin Florida (pines, oaks, woody shrubs, etc.) areaffected by G. zonaturz. Therefore, these would beaeceptable replacements. We have no definitiveinformation on the length of time for G. zonatumsurvival in the soil, but it should be measured inmultiple years, not months. It may be as long as10 to 30 years. It is certainly not less.

If an infested site must be replanted with a palm,follow these guidelines. Remove the stump and allroots from the site, and then fumigate the soil.This can be done by a professional pesticideapplicator using a legally registered fumigant forthe landscape. An example would be the productdazomet (trade name = Basamid). If the palm waslocated in a site surrounded by concrete (e.9., astreet median), remove all of the old soil, bring innew soil and then fumigate. However, none ofthese procedures guarantees the new palms willremain free of Ganoderma zonatum.

II. RESEARCH

Introduction

Although it may appear obvious that G, zonatumis a pathogen of palms in Florida, a fungalpathologist would like to have this confirmed.The term used for the process that confirmspathogenicity is "Koch's Postulates." This requiresisolating the fungus from a host with typicalsymptoms, obtaining a pure culture of the fungus,using this culture to inoculate a healthy host, andthen observing the development of typicalsymptoms and re-isolating the same fungus. \Alhilethis may be a simple task when dealing with smallannual plants, it is not simple when dealing withmature palms. Furthermore, we would like to havea systern that would consistently provide us witha method for artificially inducing the disease inhealthy palms in,:o4@er to conduct experimentsrelative to control tactics, fungal life cycle anddisease cycle.

Researchers working with the Ganoderma diseaseon oil palms in Southeast Asia have examinedpathogenicity of G. boninense using Elaeisguineensis (African oil palm) seedlings. Both naturalinoculum (Navaratnam & Leong 1965) andartificial inoculum (Hashim et al. 1993; Sariah etal..1994) were used and were successful ininducing disease. Howeveq as Hashim et al. (1993)point out, reproducing these experiments has notalways been possible.

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P A L M S G a n o d e r m a i n F l o r i d a : E l l i o t t & B r o s c h a t Volume 45(2) 2001

rjll#+r".'

F i g . 8 . T h i s m a p i l l u s t r a t e s t h o s eF lor ida count ies where Canodermabutt rot, caused by Gonodermazonatum, has been reported on palms.Information was obtained from theFlorida Department of Plant IndustryUniversity of Florida's Plant DiseaseClinics and County Extension Faculty.

Materials and Methods

Field experiments with natural inoculum andmature palms

A Syagrus romanzofflana (queen palm) withGanoderma butt rot was obtained in Palm BeachCounty, Florida, USA. This palm had basidiocarpspresent on the lower trunk that were confirmedas G. zonatum. The lower portion of the trunk wasrotted. This section (- 0.5 m) was cut into 10-15cm cross-sections with a chainsaw, and thenbroken into different size pieces (chunks, splinters,etc.) by hand. These pieces were mixed with thesawdust in a large garbage can and the lid replaced.This inoculum remained at air temperature in ashade-house for 14 days. A pure culture of thepathogen was obtained from the basidiocarp andthe wood.

A block of 28 Syagrus romanzoffiana, 20-25 yearsold, were used for the experiment. Half of thepalms were transplanted on 28 September 7994,on 6 m centers, to a site adjacent to the originalblock. Half (7) of each palm group, transplantedvs. non-transplanted, was inoculated with theinfested palm trunk material on 10 October 1994.The inoculum was divided into 14 equal portions(- 2 L volume per tree). For each inoculatedtransplanted palm, 3 equidistant holes were dug15 cm from the trunk base and about 8-10 cm

deep (until roots were contacted). The inoculumwas divided among the holes and recovered withsoil. For inoculated non-transplanted palms, theinoculation procedure was similar except that weavoided contacting or damaging any roots. Thenon-inoculated palms were not disturbed.Transplanted palms were maintained usingstandard procedures. Once they were established,no extra care (fertilizer or water) was providedthese palms or the non-transplanted palms.

Shade-house experiments with natural andartificial inoculum and seedling palms

Two experiments were initiated to determine thebest method for inducing disease by G. zonatumon seedling palms. The use of artificial and naturalinoculum was examined as well as size, amountand location of inoculum. These experiments werein i t i a ted i n June 1995 .

For Experiment I, three palm species in theseedling phase, S. romanzoffiana (queen palm),Phoenix roebelenii (pygmy date palm), and Elaeisguineenis (African oil palm), were inoculated witheither artificial or natural inoculum. Artificialinoculum was made following the method ofSariah et al. (1994). Two isolates of G. zonatumwere used, one from P. reclinata (GAN-9) and theother from S. romanzoffiana (GAN-10). Bischofiaiavanica wood was obtained and cut into 5 x 7.5

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PALMS Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

cm pieces, with the bark removed. These pieceswere sterilized for 90 min. Individual pieces werethen placed separately in heat-sealable pouchesand sterilized again for 90 min. Three days later,25 ml of sterile, cooled, but not yet solidified, maltextract agar (1 .5o/o malt extract plus 2o/o Bacto agar)was added to.each pouch. Pouches were rotatedto evenly coat the wood pieces as the agarsolidified. The next day half the pouches wereinoculated with GAN-9 and the other half withGAN-10. The G. zonatum isolates had been grownon potato dextrose agar in petri plates for 15 days.Fungal growth in each plate was cut into 5 x 5 mmpieces, and one-quarter of the pieces were addedto each pouch. Pouches were incubated at28-32C for 4 weeks. Natural inoculum was obtainedfrom a G. zonatum-infested Sabal palmetto trunk.The wood was cut into 12.5 x 25 x 50 mm pieces.

The E. guineenis and S. romanzoffiana seedlingswere transplanted from 1 L containers into 10 Lplastic containers, while the P. roebelenii palmswere transplanted into 6 L plastic containers. Thepotting substrate was 500/o pine bark, 40olo sedgepeat and 10olo sand. Roots were separated and asmuch potting mix as possible was removed priorto transplanting into containers with inoculum.All three palm species had the followingtreatments: 1) artificial inoculum infested withGAN-9, placed below the root ball; 2) aftificialinoculum infested with GAN-10, placed below theroot ball; 3) natural inoculum placed below theroot ball; 4) natural inoculum placed on top of theroot ball; 5) uninfested Bischofia wood placed ontop of root ball; 6) uninfested palm pieces placedon top of the root ball; and 7) no wood pieces.Only one piece of artificial inoculum was placedin the container. Twenty pieces of naturalinoculum were placed in each container to obtaina total inoculum volume of 15.5 cm3. There were10 replicate containers for each inoculum x palmspecies treatment.

Palms were examined at 6-month intervals forvisible symptoms or signs of the disease. After 19months, roots and crowns of P. roebelenll and S.romanzoffiana ftom each treatment wereexamined, two replicates per treatment. Aftervisual examination, roots were cut from thecrown, washed, surface sterilized, blotted dry andplaced on a semi-selective medium for G. zonatum.

For Experiment II, five different size pieces ofnatural inoculum were produced from a G.zonatum-infested Sabal palmetto trunk: a) 50 x 50x 50 mm; b) 25 x25 x 50 mm; c) 12.5 x 25 x 50mm; d) 72.5 x 25 x 25-mm; and e) 6.25 x 25 x 25mm. Only P. roebelenii palms were used with thesame potting substrate as described previously.

There were 13 treatments. Each size of inoculumwas used as either a single piece per container oras multiple pieces per container to obtain aninoculum volume of 125 cm3. The remaining 3treatments were controls with either no inoculum,1 piece of uninfested palm wood (50 x 50 x 50mm), or 8 pieces of uninfested palm wood (12.5x 25 x 25 mm for a 125 cm3 volume). There were10 replicate containers for each inoculum volumeLSize treatment.

Palms were examined at 6-month intervals forvisible symptoms or signs of disease. After 24months, inoculum pieces that were still presentwere removed, scrubbed clean, surface sterilized,blotted dry and placed on a semi-selective mediumfor G. zonatum.

Shade-house experiment with natural andartificial inoculum and mature or juvenilepalms

For Experiment III, nine palm species weretransplanted into 38 L plastic containers using thesame potting substrate as described previously.Holes, 0.5 cm diam. x 5 cm deep, were drilled intothe palm trunk or stem at the soil line (root-trunkinterface) or 15 cm above the soil surface. The fiveG. zonatum inoculum treatments were: 1) noinoculum; 2) 1 crfi of infested palm wood fromSabal causiarum; 3) I cm3 of oat kernel inoculum,made by growing G. zonatum on sterilized oatkernels for 4 weeks; 4) 1 cm: of basidiospores; and5) 1 cm3 of basidiocarp tissue. The palms specieswerc Phoenix roebelenii, Roystonea regia, DypsisIutescens, Archontophoenix qlexandrae, Veitchiaarecina, Ptychosperma elegans, Thrinax radiata,Syagrus amaret and Ravenea rivularis. The P.roebelenii palms were mature (9-yr old) and haddeveloped at least 90 cm of clear trunk. The A.alexandrae, V. arecina, and P. elegans had begun toform a woody trunk, but all other species werewithout trunk tissue at the time of inoculation.There were six replicates of each palm species xhole location x inoculum type treatment. Palmswere examined at 6 month intervals for visiblesymptoms or signs;oJ disease.

Results

Field experiments with natural inoculum

After 24 months, we observed that two of theseven inoculated, transplanted Syagrusromanzoffiana had basidiocarps present on theirtrunks. Until this point, all of the palms had asimilar appearance. Basidiocarps were removedand confirmed as G. zonatum. Pure cultures of thepathogen were obtained. These palms wereremoved with as many roots as possible and

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P A L M S Ganoderma i n F lo r i da : E l l i o t t & B roscha t Volume 45(2) 2001

replaced with S. romanzoffiana juveniles. To date,no other palms in this experiment have died orproduced basidiocarps of G. zonatum.

Shade-house experiments

No palm was killed by G. zonatum or ever exhibitedsymptoms of the disease in any of the threeexperiments. The fungus was not detected in theroots examined in Experiment I, nor was itdetected in the inoculum examined in ExperimentII. We observed that the center of the Bischofiawood pieces used in Experiment I were completelydegraded leaving a hollow core.

Discussion

The only time Ganoderma butt rot was inducedwas when natural inoculum was used on maturepalms. Even then, only 2 of 14 inoculated palmsexhibited symptoms and signs (basidiocarps) ofthe fungus. Howeveq inoculation of field-grownpalms is not a good system for completing Koch'spostulates. First, working with palms with talltrunks is difficult and expensive. Second, there isno way of definitely determining if these palmswere already infected with G. zonatum. Ourattempts to use seedling palms (i.e., palms withoutwoody trunk tissue) for pathogenicity assays werenot successful, whether we used artificial or naturalinoculum. We also saw no evidence of root rot orroot infection as has been observed with oil nalmseedlings inoculated with G. boniiense(Navaratnam & Leong 1965; Hashim et al. 1993;Sariah et al. 1994). Even the experiments thatinoculated mature Phoenix roebelenii and variousspecies of juvenile palms with different forms ofinoculum placed directly into the trunks wereunsuccessful in inducing the disease. However,the observed infection of clumping palms (e.g.,Dypsis lutescens) that have mature canes removed,the infection of two of the seven transDlantedSyagrus romanzoffiana, and the pattern of diseasedevelopment within the trunk suggests that G.zonatum probably enters palm trunks via deadpalm tissue (dead roots or palm stumps) as asaprophyte and spreads from there into adjacentliving woody trunk tissue. Additional experimentsare planned to test this hypothesis.

Acknowledgments

We thank S. Thor and E. A. Des Jardin for field,shade-house and laboratory assistance. Thisresearch was supported, in part, by grants from thePalm Beach Gardens Mall and the Royal PalmChapter of the Florida Nursery GrowersAssociation. Florida Agricultural ExperimentStation Journal Series no. R-07860.

Lirturunp Crrpn

AoRsxavrc, J.E. aNn R.L. GrrnrnrsoN. 1988.Basidiospores, pilocystidia, and other basidio-

carp characters in several species of theGanoderma lucidum complex. Mycologia 80:493-507.

An.a.sx.rvEc, J.E. nNn R.L. G[srRrsoN. 1989. Culturalstudies of four North American species in theGanodeftna lucidum complex with comparisonsto G. Iucidum and G. tsugae. MycologicalResearch 92: 182-797.

Ao.asxlvrc, J.E., R.A. BrlucHErrp, AND R.L.GIrsERrsoN. 1991. Decay of date palm wood bywhite-rot and brown-rot fungi. CanadianJournal of Botany 69:615-629.

Ar.rmRrJn., S.A., K.R. LaNcooN,J.W. Krvnnoucu, N.E.Er-GHorr axo C. WnHr.sunc. 1994. Diseases anddisorders of plants in Florida. Bulletin No. 14,Division of Plant Industry, Florida Departmentof Agriculture and Consumer Services,Gainesville, Florida, USA.

CHIrns, J.F.L. ml E. WEsr. 1953. Butt rot of queenpalms in Florida associated with Ganodermasulcatum. Plant Disease Reporter 37: 632-633.

GrmEnrsox, R.L., .Lun L. RyvennpN. 1986. NorthAmerican Polypores. Volume l. Abortiporus -Lindtneria. Fungiflora, Oslo, Norway.

HnsHrlr, K., L.T. KwpE aNu A.R.A. Razax. 1993.Pathogenicity of Ganodermq boninense Pat. onoil palm seedlings. In Proceedings 1991 PORIMInternational Palm Oil Conference - Agriculture(Module l), 478-423. Palm Oil Research Instituteof Malaysia, Selangor, Malaysia.

MIrrEn, R.N.G., M. Hor"oEnNESS, P.D. BnrncE, G.F.CuuNc aNo M.H. Zarapra. 1999. Genetic diversityof Ganoderma in o11 palm plantings. PlantPathology 48: 595-603.

Moucarvo, J.-M., H.-H. WaNc aNn R.-S. HsEu. 1995.Phylogenic relationships in Ganoderma inferredfrom the internal transcribed spacers and 25Sribosomal DNA sequences. Mycologia 87:223-238.

MuRRrLr, W.A. 1902. The polyporaceae of NorthAmerica. I. The genus Ganodermq. TorreyBotanical Club Bulletin 29: 599-608.

Munnnl, W.A. 1908. Ganoderma. North AmericanF lo ra 9 : 1 18 -123 .

N.qvlnarNevr, S.J. eNo C.K. LEoNc. 1965. Rootinoculation of oil palm seedlings withGanoderma sp. Plant Disease Reporter 49:101 1*1013.

SanraH, M., M.Z HussrN, R.N.G. MrrrsR AND M.HoroEnNlss. 1994. Pathogenicity of Ganodermaboninense tested by inoculation of oil palmseedlings. Plant Pathology 43: 507-5 10.

ScHuernl, T.S., W.N DrxoN, AND R. Lntnv. 1997.Florida's most wanted top 10 diseases.Ornamental Outlook 6(8): 10.

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