FETGAL ASSOCIATIONS IN TEE BUILE-UP AiTR DECLINE OF CRETOCOCCIIS FAGISUGA WPULATIOI~S~ David ~onsdale~ Abstract.--The fungal flora of Czyptoooccus fagisuga colonies on Faaw svZvatica bark included the entomo~enous- species ve'ertic2~2i& Zecmrii wherever infestation was or had been very hea-?y. This fungus seemed to accelerate insect mortality in vitro. CZadosporium cZadosporioides was present at all stages of insect colony development and, with other fungi, caused blackening of the wax secretion. This blackening has been recognised as an indication that the colonies contain many dead The great economic importance of many insects and are probably declining. It appears species of scale insect has long made them a that blackening is also related to the degree of major object of man's efforts in biological wetting from rain, in relation both to water control. Their colonial, sessile habit and runnels on stems and to varying weather condi- predictable occurrence in space and time tions. favours the use of predators and parasites. As the primary agent in the beech hark disease The microflora of infested bark merits complex (Ehrlich 1934, Parker 1974) study not only for the possible detection of Cqptococcus fagisuga Lind. seems a worthwhile entomogenom fungi but also for elucidating the target for biocontrol, and yet it has been pre-Lnfection ecolom of bark-invading species studied very little to this end. of Nectria. It is of particular interest that C. fagisuga is consistently associated with Several predators of C. fagisuga are Nech'ia coccinea Fries. or with If. coccinea var. known. These include the coccinellids faginata Lobn, Watson & Avers in the beeeh hsrk Ecochonius and Chitocorms spp., the cecidomyid disease complex. Some Nectria spp. are known as fly kstodipZosis sp. and various chrysopids parasites of scale insects (Petch 1921) and and hemerobiids, but their importance in although this does not necessarily imply that the regulating populations of the insect is relationship between C, fagisuga and N. coccinea thought to be limited (D. Wainhouse pers. involves parasitism, it does seem likely thst ccmrm.). No insect parasites of C. fagisuga N. coccinea is ecologically favoured within the are known. microflora of Cryptococcw-infested beech bark (Lousdale and Sherriff, these Proc.) As far as microbial control agents are . concerned. only one fungus has been reported The obSectives of the nresent work were to to exert some influence over C. fagisuga. investigate the fungal flori associated with This is AscorEichaewa mgosa which forms a different phases in the growth and decline of tough, black stroma at the bark surface, thus C. fagisuga populations and to examine the deterring the insertion of the insect's possibility that the insect is susceptible stylets s ousto on 1976; Houston, Parker and to attack by entomogenous fungi. Lonsaale 1979). No association between C. fagisuga and any entomogenous mgus has been reported, although- it seems clear that a microflora is associated with rhe inscct colonies, as evidenced by the commonly observed blackening of the white wax secreticn. Detection of fungi associated with C. fa&uga 1 Paper presented at the IUFRO Beech Bark Disease Working Party Conference, Hamden, Throughout this work several types of CT. USA. 27 Sep to 7 Oct 1982. C. fagisuga colony development were defined so Forest Pathologist, Forestry Commission as to represent different phases of build-up and Research Station, Alice Aolt Lodge, Farnham, decline. In defining these types, the colonies Surrey, England. were classified by size and by the extent to 99 which dead insect bodies and blackened wax had On the basis of these observations, a accumulated. In this way, colonies are series of platings of individual live second described as small or large and new or old. instars was set up on 0.01% YEA. These insects Where appropriate, a score class for the were obtained from trees at Alice Holt Forest, overall insect density on the stems was used -shire and were removed by dissection of in combination with the classification of colonies in sib. Five different types of colony type. insect infestation were recognised in this survey as shorn in Table 1, which includes all Preliminary observations indicated that Pungi which were isolated from 10%or more of the most frequently occurring fungi on insects in at least one of the infestation C. fagisuga-infested bark were present at all types. These data show that V. Zeoanii was forest sites studied, and that all heavily abundantly present on insects where the infested trees carried populations of these infestation either was or had been in the form fungi. Three fungi commonly isolated were of extensive cwer on the bark. Nectria Vertia'ttiwn Zeumii Viegas which is an viridescens and C. cZadospoptPtddsawere present entomo~enouss~ecies (Petch 1948).-. a on insects from all infestation types. synne&us ~&nwniwn (the imperfect stage Fusapiwn lateritiwn Nees. seemed to be associa- of Nech.ia viridescols Booth) and ted with old, declined infestations, while CZadosporim ~Zadosporioides(R-es .) de Vries Mucor sp. was largely confined to small, which seemed primarily responsible for recently established colonies. The fungi which blackening of the wax secretion. occurred on fewer than 10%of the insects in Table 1.--Fungi frequently isolated from live C. fagisuga adults and larvae plated on 0.01% yFAb (see text for other species) Percentage of insects yielding each fungus na B C D E WgUS (discrete) (discrete) (residual) (whitewashed) (residual ( 'BBD tree' ) Vertici ZZiwn teumii o o 56 69 74 Nectria viridescens 31 21 8 3 14 Cladosporiwn spp .C 25 36 16 3 6 Fusariwn loteritiwn o o 16 o 9 Mucor sp. 38 0 4 0 0 PmiciZZ&w sp. 6 0 4 19 3 a: Arller descriptions of infestation types are: A, small isolated (t2mm mean diameter), well separttted colonies with little or no black wax; B, large (>5mmean diameter), well separated colonies; C, low current infestation on trees with previously heavy infestation; D, stem continuously colonised over large areas; E, infestation pattern as for 'C' but bark with inactive, callused Nectria lesions. b: yeast extract agar. c: almost entirely C. ckzdosporioides. any infestation tme included the followinn- incubation under humid conditions at room hypbolqycetes: l%ihodenur viride, temperature. Only V. kCUnii was scored in Zh'chotheuim sp., hsan'm menaceum, this way, although some of the fungi named Rmmchlot+dim subulam. Altermaria alte~nata. above were also observed by this method, StemphyZium botqosum, ehtinu~asp., Dip20cli& together with several Acz%?mniwn spp. and a sp., Pyrenochasta sp. (an un-named species), Spomthrix sp. The discs were taken from lipicocnm, sp. MmiZia sp. StachyZidium sp. trees with different overall infestation DzctpZZa sp. and at least three non- densities at Ween Elizabeth and Marden sporulating fungi. Yeasts, especially Forests in Hampshire and West Sussex Aweolusidiwn sp., were frequently present oh respectively. The data, which are shown in insects from all colony types other than Table 2, are based on infestation classes 'whitmashed', and bacteria were present (scores 0 to 5) of the type used by nouston, throughout the samples. Nectria cocuiwa was Parker and Lonsdale (19791, classes 0 and 1 not detected on individual insects, but was being excluded from the present study. Within isolated from mass platings of insects each of these classes variation occurred in together with their wax. the apparent age of the colonies, and a classification of old and new infestation was Of the fungi detected, V. Zecmii was used. 'Old' infestations were recoegised by the only species known to include entomogen- the presence of substantial blackening and ous strains, and a further study was carried erosion of the wax. out to gain further information on the extent to which this fungus was dependent on insect These bark disc incubations provided density. This study involved the counting of confirmation that V. Zeconii was dependent on insect colonies from which V. Zecrmii grew insect density and they also showed-an effect during the incubation of 25mm discs of of age of infestation. On new infestations, C. fagisuga-infested bark. The fungus was only class 5 yielded a substantial score for readily detectable after three or four days' V. lecmii. However, in older infestations Table 2.--Outgrowth of V. Zeamii from incubated bark eiscs in July 1982 Mean percentage of individual wax masses yielding V. Zeomrii Infestation class 2(light) 3boderate) h(heaq.) 5(very heaq.) Colony tme smalla colonies 6 large' infestation Abs colonies . smslla 'Old' colonies largeb infestation Abs colonies . a <3mm mean diameter b 93nnn mean diameter. Abs. indicates large colonies absent in class 2 infestation. For clarity, the above data are pooled for the two forest sites and expressed as percentages. Due to heterogeneity between sites, statistical significance, based on x2tests of the actual counts was estimated separately for each site and for old and new infestation with the omission of poorly replicated classes. The effect of infestation class was highly significant (p c0.01 to p c0.001) except for small colonies on new infestations at &den Forest (0.10 ep >0.05). the fungus was substantially present on carried out simply by scoring the production of colonies with a score class of three to four. external growth of the fungi on the insect This occurred whether or not most of the old bodies afier death (Table 4). These date show colony material had been washed away by rain. that PaeciZonp/ces f&nosus was by far the most effective colonist of the insects. Plate 1.-- Conidiophores of V. kcmii growing from C. fagisuga colonies (x 100) Fungal inoculations of C. fa.qi8wa in Vitro In all the inoculation studies, indivi- dual live first instars were placed on sterilised filter paper in hlnnidity chambers (95% R.A.) at 20'~.