WITH FRUTESCENS FUNGI ASSOCIATED BORRICHIA AND ENDOPHYTES GALLS (ASTERACEAE): INSECT Amy Haddock Keagy,and Diane Te Peter D.Stiling Strake, Department of Biology University of South Florida Tompa, 33620-5200. Florida U.S.A. ABSTRACT DC, This report compares fungi found in galled and non-galled plants of Borrichia Jrutescens (L.) & Asteraceae. Fungi were observed in the plant galls of the midge, Asphondylia borhchiae Rossi Strong. A variety of endophytic fungi were cultured from the apical meristems, stems and leaves of galled and non-galled plants collected from several coastal sites in Florida. Fifteen percent of midges examined in - 60) carried fungal spores of Alternaria sp. RESUMEN compara hongos cncontrados en plantas con sin agallas de Borrichia Jrutcscens Esce articulo los y & (LJ DC. Asteraceae. Se observaron hongos en las agallas de Asphondylia horrichiac Rossi Strong. Se cultivo una variedad de hogo cndof itico de los meristemos apicales, tallos y hojas de plantas con y sin agallas colectadas en varios lugares costeros de Florida. El quince por ciento dc las agallas examinadas (n = 60) tenian esporas de Altcrnayia sp. INTRODUCTION / marsh communities Gulf Asteraceae often found bordering the of the of salt Mexico and Atlantic Ocean. Insect galls on this host species are initiated in the & meristem by midge, Asphondylia horrichiae Rossi Strong (1990). apical a These galls are also impacted by four specific parasitoid wasps iGaleopsomyia haemon Walker, Rileya cecidomyiae Ashmead, Tenuipetiolus teredon Walker, and Torymusumhilicatus Gahan). Individuals from these parasites lay their eggs inside the gall with A. horrichiae. The parasitoids devour the developing midges and population horrichiae (Rossi 1992). significantly affect levels of A. et al. Several biological studies on this plant midge system elaborate on the lite his- & mcludmg tory and ecological details parasitism rates (Rossi Strong 1990; & many As Stiling et al 1992; Stihng 1994; Rossi Stilmg 1998; Rossi et al. 1999). in becomes insect the tissue surrounding the developing larvae eventually galls, of chambers layered with fungal growth purportedly providing nutrition full knowledge the developing larvae (Gagne 1989). Thus, of the fungi associ- for ated with both the galled and non-galled plants needed to gain insight into is host-parasite relationship. this r Many known endophytes which predomi- harbor microbial plants are to tern. SIDA22(1):755-763.2006 756 BRILORG/SIDA 22(1) nantly These organisms arc fungi. live internally in plant tissue. Stone et al. endoph (2000) suggest that these y tes secondarily invade insect galls. Batra and Lichwardt grown (1963) nidicate that "gall fungi are airborne, in a variety of and substrata are not species specific.^' Other nivestigators of nisect galls sus- pect that spores of fungi are accidentally collected from leaves and leaf litter & by the insects (Borkent Bissctt 1965), Haridass (1987) suggests that con- fecal means tamniation another whereby is possible of spore dispersal fungi are in- troduced into the plant tissue. Gagne (1989) reports that midges feed by suck- ing on the hyphae and states, "fungi are evidently obligatory in all galls...and are food of those gall midges." Batra and Lichwardt (1963) alternatively indi- cate that is difficult to determine whether midges actually feed on the hy- it phae or the plant tissue degraded by the fungi. we examined number In this study a of midge galls of Asphondylia from borrichiae Borrich iafrutcsccnsirom several Florida the presence sites for or absence ol lungi. In addition plant apices, sterns, and leaf samples of galled and non-galled plants were cultured for fungi. Finally, a small population of midges was surveyed for associated fungal spores. MATERIALS AND METHODS — Sites for Collections, Borrichiafrutcscens and stem samples were galls, leaf m from collected the following locations from 1993-1995 Florida: Site #1-Merritt Island National Wildlife Refuge, Titusville (Brevard County). Mayport Site #2-Little Jetties Park, (Duval County). Site #3-Ft. DeSoto State Park, Petersburg (Pinellas County) St. ^4-Upper Tampa Site Bay Park (Hillsborough County). #5-Delnor-Wiggins Site State Park, Naples (Collier County). — Culture Protocol. In May, 1995, five samples each of apex, stem, and leaf tissue were collected from galled and non-galled plants from and Sites All col- 1, 2, 3. m lected plant samples were placed individual, sterile plastic bags, refrigerated, and returned to the laboratory within three days. Excised tissue from apices, stem and both and leaf tissue of galled non-galled plants tissues were rinsed in running water lor 5 min, then each tissue type was placed separate in flasks mm and 95% rinsed with ethanol for 2 min. This was followed by 30 shake a 20% rinse in bleach solution. All tissues were held in flasks of sterile distilled water (1- 2h) while tissues were prepared incubation. Three from each for slices m tissue from each site were submerged Potato Dextrose Agar (PDA) and V-8 m Agar Juice (Difco, Detroit, Ml). These cultures were placed dark incubator a 32^C PDA seven from at for days. Isolates successful cultures placed on w^ere slants and held at ambient temperature until sporulation after which identifi- cations were made. TE STRAKE ET FUNGI ASSOCIATED WITH BORRICHIA FRUTESCENS 757 AL., — In Siiu Fungal Studies. In September, 1995, ten stems were collected ran- domly from galled and non-galled plants of B.frutescens from four sites (# 1- cm Each stem was cut into segments. Using the protocol established by 4). 1.5 Hignight et (1993) stems were cleared and then all segments were micro- al. examined scopically the presence of endophytes. (or — Whole Direct Observations of Insects. insects of AspJio?i-:Iy!]a horrichiae were collected as they emerged from galls of Borrichiajrutesccns, (n - 60), Iva imhricata Walter (n - 20), and IvaJrutesccnsL. (n = 5) and preserved in vials of 95% ethanol. All msect samples were examined for the presence or absence of fungal spores on the surface using light microscopy. AND RESULTS DISCUSSION chambers Previous dissection of the Borrichiafrutescens gdlls revealed larval Within white mycelium could be observed and fungal these this (pers. obs.). growth was arrested until the larvae became fully developed. Then the myce- mat became and denser, darker, quite distinct (Fig. lial 1). Midge galls observed in the Asteraceae, and fungi isolated Irom those galls Aecidium are listed in Table In addition, Farr et (1989) note the occurrence of al. 1. horrichiae, Puccinia mi and Cercosporidium sp. on stems and leaves of B. rifica, many frutescens. Although plants have fungal endophytes (Clay et 1985; Clay al. known were from 1990; Carroll 1988), prior to this they not B.frutescens. and Preliminary studies (1993-1994) including direct observations excis- ing and culturing of a range of ages of gall tissue from B.frutescens from all five two sites consistently yielded a large variety of fungi including types of sterile made mycelium (data not shown). Similar observations have been in other stud- ies of midge galls. Bissett and Borkent (1988) report that some midges from the Lasiopterini and Asphondylidi inoculate galls with a variety of mitosporic fungi and use them as a food source. Stone et (2000) observed that galls on Dou- al. (Pseudotsuga menziesiO support heavy fungal growth which, he sug- glas-fir may Wilson be plant pathogens in addition to providing insect nutrition. gests, may (1995) proposes that fungi in galls be saprobes or inquilines (organisms maker inhabiting insect galls not parasitizing the gall but otherwise utilizing the gall tissue lor food). and To determine endophytic fungi could be observed microscopically if method Hignight preliminary study was conducted using the of et al. (1993), a in May 1995 using five steims each from galled, non-galled, flowering non-galled from and flowering galled plants of B.frutescens collected Site #3, Ft, Desoto. 100% This mini-study revealed that endophytic fungi were present in of the example samples from plants with galls appearing similar to the seen in Figure The remaining non-galled stems were heavily colonized with iungi. This less 2. quantify endophytic fungi qualitative observation led to additional studies to 758 BRIIORG/SIDA 22(1) Fig. ].Borrichia frutescens. Cross section through a gall showing the layer of the fungal mycelium. We involved in the stems of galled and non-galled plants. observed a consistent occurrence of endophytic fungi in sampled stem segments from four diverse all Florida locations (Sites 1-4). In non-galled plants these occurrences range all 64% from 20 and to in galled plants 30 to 64%. This suggests httle influence of the role of the midge. But one might argue that the fungal endophytes in galls On grow out, invading other plant tissue. the other hand, depending on the time- may frame, endophytic fungi grow into the gall or exist in the pre-gall tissue and mycof contribute to the lora of the aging Either way, fungal endophytes gall. m are extant Bjrutesccns. and In the apices, stems, leaves of both galled and non-galled plants from and among Sites 3 several fungal taxa occur (Table Included the 1, 2, 2). isolates among are several types of sterile mycelium. These were found all tissue types. was among Altcrnaria found Acrcmonium sp. also all tissue types (Table strictum, 2). 759 TE STRAKE ET AL., FUNGI ASSOCIATED WITH BORRICHIA FRUTESCENS Table Fungi reported In gall midge associations in Asteraceae. 1 . Reference Fungi midge Host Gall Sderotium Weis 1982;Batra 1964 Asteromyio carbonifero Solidago canadensis asteris Macrophoma Borkent&Bissett 1985 modesta mollis sp. A. tumiflca,A. S. carbonifero A. 1964 Rhytisma Batra Aster asteris A.carbonifero sp. 1964 Batra sempervirens bifrons A.corbonifera R. S. 1964 Batra S.lanceolata R.solidaginis A.carbonifera Chaetoniium globosum Batra 963 Ratibida columnifera A.rotibidoe 1 1963 Aureobasidium Batra R.columnifera A.ratibidae pullulans Plectosporium R.columnifera A.ratibidae tabacinum (asCephalosporium Batra 1963 ciferrii) 1963 Batra A.carbonifera S.graminifolia Alternaria sp, 1963 Trichoderma Batra simulans Helianthussp. viride Bucculatrix 1963 Batra simulans Heiianthus sp. Aspergillus sp. B. roseum 1963 Trichothecium Batra Helianthus simulans sp. B. 1963 Batra Heiianthus Penicillium spp. simulans sp. B. J 963 Stemphylium Batra Helianthus sp. simulans sp. B. 1963 simulans Heiianthus sp. Botrytis cinerea Batra B. more irregular distribut^^^^ Bipolarfssp.,andVerticzniumlecaniidisplayed from sampled. Other species of these genera have been the plant tissues sites & White (Bacon 2000). endophytic fungal hterature reported in the abdomen) midges The mycangia pockets adjoining the of in (small special known be with fungal spores that are thought to be in- wild the are to filled examined newly emerged midges in serted with the eggs at oviposition. In the were no spores were seen in the mycangia. Spores of Alternaria sp. this study, 15% midges emerging from Borrichiafrutescens seen on the surfaces of of the 40% and = 10% the midges from Iva imhricata (n-20); of these galls (n 60); of / / Wh seems that the diversity of fungi seen in these fungal spores oviposition, at it midge and host plant tissue are sourced by other means, possibly includ- galls some ing of their parasitoids. We endophy appear become denser in the plant observed that fungal tes to m May preliminary study) and September revealing between (seen the tissues m were hyphae and pigmentation The aging galls this study increasing (Fig. 2). became denser and darker with pigments seen include fungal contents that to and both darkly pigmented, were consistently Alternaria Bipolaris (Fig. sp. sp., 1). 760 BRIT.ORG/SIDA 22(1) Table 2. Percent occurrence of fungi isolated from apical,bteni and leaf tissues isolated from Borrichia and frutescens: galled non-galled plants. Merritt Island Mayport Desoto Ft non-gall gall gall non-gall gall non-gall mycelia Sterile Apex - 333 66.7 13.3 40 Stem 20 33,3 20 26.7 Leaf 6.7 40 .7 13.3 6.7 Alternaria sp. Apex 13.3 13,3 Stem 6.7 Leaf ?0 6. 33.3 6.7 13.3 Verticillium lecanii (A.Zimmerm.) Viegas Apex Stem 6.7 Leaf 20 20 Bipolans sp. Apex Stem 6.7 Leaf 6. Acremonium Gams strlctum W. Apex Stem 13 20 Leaf Traaen f7u/77/co/a gr/sea Apex Stem 6.7 Leaf Monocillium indicum S.B.Saksena Apex Stem 6,7 Leaf Peniclllium sp. Apex 6 Stem Leaf Fusarium sp Apex Stem 6.7 Leaf STRAKE AL, FUNGI ASSOCIATED WITH BORRICHIA FRUTESCENS 761 TE ET ^ ^ f J ^ fe ^i .:^ .r*^. .^ .^v" Hyphae endophytes stem hQ.I.Borrichiafrutescens. of in pith tissue. Shaw reviewed from and non-galled plant (1992) several isolated galled tissue. pigmented darkly studies indicating that fungivores consistently favored litter seems Altcrnaria and Bipolaris could play a fungi. Interestingly, that sp. sp. it system. nutritional role in this gall study we establish the presence of entophytic fungi in insect galls. In this Endophytic fungi were cultured from tissues of galled and non-galled plants These Both these plant types carry several fungal taxa. Borrichiafrutescens. of of common observations are to all sites, but sample sizes are too limited to discern exammed on 15% major Fungal spores were directly observed of differences. may midges. This suggests that the midges play a role in fungal dispersal to gall tissue as proposed by Batra and Lichtwardt (1963). ACKNOWLEDGMENTS comments and Hennen, The authors greatly appreciate the suggestions of Joe anonymous and one Erica Cline, reviewer. REFERENCES New endophytes. M. Dekker. York Bacon, C. W. and J.R White Jr. (eds.) 2000. Microbial Inc.. 762 BRIT.ORG/SIDA 22(1) Batra, L,R. 1964. Insect-fungus blister galls of Solidago and Kansas Entomol.Soc. /\sterJ. 37:227-234. and some Batra, L.R. R.W. Lichtwardt. 1963. Association of fungi with insect Kansas galls. J. Entomol.Soc. 36:262-278. BissETTj.and Ambrosia A. Borkent. 1988. galls:The significance of fungal nutrition the in evolution of the Cecidomyiidae (Diptera). Pirozynski and Hawksworth, Co- In: K. D. eds. 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