PHYTOPHTHORA AND PYTHIUM SPP. FROM PINE PLANTATIONS IN

By E. M. DAVISON*t and M. BUMBIERIS*

[Manuscript received 7 August 1972]

Abstract A survey was carried out during the winter of 1971 to determine whether Phytophthora and Pythium spp. are present in South Australian pine plantations. Phytophthora cryptogea was frequently isolated from Kuitpo forest reserve in the hills, but Phytophthora cinnamomi was isolated from only one tree in a pine seed orchard. Pythium anandrum and pythium mastophorum, never previously isolated in South Australia, were frequently found in various forest plantations.

I. INTRODUCTION Interest in Phytophthora spp. as root pathogens of forest trees has developed in Australia and New Zealand since Newhook (1959) showed that many could be isolated from dying pines in New Zealand, and Oxenham and Winks (1963) isolated P. cinnamomi Rands and P. boehmeriae Sawada from dying pines in Queensland. P. cinnamomi has been found to be widely distributed since it was first isolated from jarrah dieback areas in Western Australia (podger et al. 1965), and it is now regarded as a major pathogen of both indigenous and introduced woody plants (Podger and Ashton 1970). P. cinnamomi was first isolated in South Australia in 1969 in association with plants belonging to the families Proteaceae, Myrtaceae, and Ericaceae (Davison 1972). A survey was carried out during 1971 to determine whether Phytophthora spp. are present in South Australian pine plantations. These consist predominantly of Pinus radiata D. Don. with small areas of Pinus pinaster Ait. and are situated in three main areas: the south-eastern part of South Australia, the ranges near Adelaide, and the mid-north section of the State. The two cooler areas of and the south-east of the State receive considerably more rainfall than the mid-north of the State as illustrated by the average annual rainfall figures published by the Bureau of Meteorology: Gumeracha (near Mount Crawford and Kersbrook forest reserves in the Adelaide hills), average for 81 years-32 ·62 in.; Mount Gambier (in the south-east of South Australia), average for 89 years-30· 54 in.; and Bundaleer

... Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, S.A. 5064. t Present address: c/- Dr. P. J. N. Davison, Mullard Space Science Laboratory, Holmbury St. Mary, Dorking, Surrey, England.

Aust. J. bioI. Sci., 1973, 26, 163-9 164 E. M. DAVISON AND M. BUMBIERIS

forest in the mid-north, average for 93 years-2l·62 in. Trees in the Adelaide hills forests are often planted on steeply sloping hillsides, with numerous creeks present in some areas. Although the hillsides in the other two plantation areas are not so steep, low-lying sites become very wet during winter, especially in the south-eastern part of the State. Sampling for species of Phytophthora and Pythium was carried out both in the Adelaide hills and the south-eastern plantations on sites which had a history of tree death and low productivity. These were often situated on sandy soil overlying clay, liable to waterlogging in winter and drying out in summer. Forest nurseries were also included in the survey because transplanting of infected seedlings could be a method of spreading a potentially dangerous pathogen (Oxenham and Winks 1963). Because of the generally drier conditions the mid-north plantations were not included in the survey.

II. METHODS Soil samples containing fine roots were taken at a distance of about 50 cm and a depth of about 30 cm from two sides of trunks of pine trees showing foliage symptoms such as little leaf and premature needle fall. Samples were also collected from near recently dead trees and stumps of salvaged trees. Each sample (about 250 g) was divided in two parts and placed in plastic con­ tainers (10 cm in diameter and 6 cm deep). A green pear, of either the cultivar Josephine or Packham Triumph, was placed in each container, and enough rainwater was added to cover the soil to a depth of about 2 cm. This "baiting" method, originally used by McIntosh (1964), was at first supplemented by the baiting method described by Chee and Newhook (1965) using young seedlings of Lupinus angustifolius L. or Pinus radiata. As the pear-baiting method appeared to give the more consistent results and is simple it was used in preference to other baiting methods. Areas of rot appeared on the pears after 3-6 days. Pears were then washed in tap water, dried, and passed quickly through a flame. Small pieces of about 3 mm2 were taken from the edges of rotted areas and plated out on cornmeal agar. Small pieces of pine roots showing symptoms of rot were sometimes surface sterilized in 1 % sodium hypochlorite for 30 s and then plated on the selective 3P medium (Eckert and Tsao 1962). Phytophthora species were identified using Waterhouse's (1963) key. Cultures grown on V-8 agar (200 ml Campbell's V-8 juice, 800 ml water, 2 g CaCOa, and 15 g agar), and submerged either in 1 % non-sterile soil extract or rainwater were used for production of sporangia or oogonia or both. When oogonia were not produced in single cultures, colonies were paired with isolates of other strains or species. The tentatively identified cultures were later compared with known isolates from the Commonwealth Mycological Institute. Pythium spp. were identified using the keys of Middleton (1943) and Waterhouse (1968). An isolate of Pythium at first thought to resemble Phytophthora stellata Shanor was later identified as Pythium anandrum Drechsler by Dr. D. J. Stamps of the Commonwealth Mycological Institute, although it differs in some respects from the original description. The differences are production of antheridia and more mammiform oogonial spines.

III. EXAMINATION OF FOREST NURSERIES Table 1 shows that Phytophthora spp. were isolated from two forest nurseries: Belair nursery, where no pine seedlings are grown for forest plantations, and Gorge Road nursery, where P. megasperma Drechsler var. sojae Hildebrand was found in a very wet spot usually not used for raising pines. As no Phytophthora spp. were found in association with pine seedlings in forest nurseries in this or previous surveys PHYTOPHTHORA AND PYTHIUM SPP. FROM PINE PLANTATIONS 165

(Bumbieris, unpublished data), the possibility of these fungi having been carried from nurseries to plantation sites seems unlikely.

TABLE 1 PHYTOPHTHORA SPP. ISOLATED FROM FOREST NURSERIES

Location of nursery Source or host Phytophthora spp.

Adelaide hills Belair Soil citricola cryptogea Prostanthera sieberi Benth. nicotianae var. parasitica Cudlee Creek Pinus radiata Gorge Road Soil megasperma var. sojae Kersbrook Kuitpo } P. radiata Second Valley South-east Mount Gambier Headquarters Myora No.2 Penola Headquarters } P ,"du"" Penola No.2

IV. EXAMINATION OF FOREST PLANTATIONS Results of the survey of forest areas in the Adelaide hills (Table 2) show that out of 94 trees sampled at Kuitpo forest reserve 20 yielded Phytophthora cryptogea P.ethybridge & Lafferty, and two yielded P. megasperma var. sojae. One tree at Mount Bold seed orchard, which is a section of Kuitpo forest reserve, yielded P. cinnamomi; P. cryptogea was also present in the seed orchard. Why P. cryptogea was isolated with high frequency from the Kuitpo forest reserve but not other areas is not yet clear. It is possible that more extensive sampling would reveal the presence of this fungus in other forests, especially in the Adelaide hills. P. cryptogea was most frequently isolated from wet creek-bed sites, many of which were submerged in water during the very wet winter of 1971. This condition was not encountered at sampling sites in the Kersbrook and Mount Crawford plantations, as water at these sites drained faster either because of steeper slopes or different soil structure. The very wet conditions combined with low winter tempera­ ture may have induced abundant zoospore production in soil-root samples later kept at room temperature facilitating recovery of the fungus. The only Phytophthora isolated from samples in the south-eastern forests (Table 3) was P. megasperma var. sojae. Nearly complete failure to isolate Phyto­ phthora from the south-eastern forests may indicate that they are rare, or possibly be explained by the comparatively small number of samples from each site, the greatest number of trees sampled being 37 at Mount Gambier forest reserve as compared with 94 at Kuitpo. Further work is necessary to distinguish between these two possibilities. Phytophthora spp. were never isolated from rotting root pieces plated on 3P medium. Various Pythium spp., in addition to other fungi, have previously been recorded in association with pine seedlings in South Australian forest nurseries (Vaartaja and 166 E. M. DAVISON AND M. BUMBIERIS

TABLE 2 ISOLATIONS FROM PINE PLANTATIONS IN ADELAIDE IDLLS

Age of No. of Soil tree trees Phytophthora spp. Pythium spp. type* (yr) sampled

Mount Bold seed orchard 12 cinnamomi (1)t irregulare (2) cryptogea (1) mamilla tum (1) Kuitpo forest reserve 38 A-C 5 cryptogea (4) 44 Es 1 cryptogea (1) mastophorum (1) 39 Mfs 10 cryptogea (2) anandrum (2) 35 Mfs 5 cryptogea (4) mastophorum (2) 31 A-C 1 cryptogea (1) mastophorum (1) 9 1 cryptogea (1) anandrum (1) 9 Mfs 15 megasperma var. sojae (2) anandrum (3) 8 Kgsl 21 cryptogea (5) mamillatum (1) mastophorum (1) anandrum (3) 20t cryptogea (2) anandrum (1) irregulare (1) 49 KHS 2 irregulare (1) mamillatum (1) mastophorum (1) 33 Mys 3 mastophorum (1) 23 Mfs 5 anandrum (2) Mfs§ 5§ irregulare (4) mamillatum (3) mastophorum (2) anandrum (1) Mount Crawford forest reserve 16 10 mamillatum (1) 14 4 mamillatum (1) 13 8 mamillatum (2) anandrum (2) 9 8 acanthicum (1) irregulare (1) mamillatum (1) anandrum (1) 6 7 anandrum (1) mamillatum (1) Kersbrook forest reserve 22 4 mamillatum (3) anandrum (1) 10 5 mamillatum (1) mastophorum (2) * A-C, alluvial complex; Es, sand; Kgsl, Kuitpo gravelly sandy loam; KHS, Knots hill sand; Mfs, Meadows fine sand; Mys, Myponga sand. Classification of Rix and Hutton (1953). t Number of trees from which the species was isolated. t Pinus pinaster; all other trees were P. radiata. § Old nursery site. PHYTOPHTHORA AND PYTHIUM SPP. FROM PINE PLANTATIONS 167

Bumbieris 1964; Vaartaja 1967). Of these P. irregulare Buisman, P. mamillatum Meurs, and P. acanthicum Drechsler were also isolated from the forest reserves in the Adelaide hills. On the other hand, P. anandrum and P. mastophorum Drechsler have never been found in forest nurseries or in association with other plants in South Australia, and their importance with regard to pines requires further investigation.

TABLE 3 ISOLATIONS FROM PINE PLANTATIONS IN THE SOUTH-EAST

Ageof Soil No. of tree t * trees Phytophthora spp. pythium spp. (yr) ype sampled

Penola forest reserve 9 Kas 16 megasperma irregulare (4) var. sojae (1)t mamillatum (1) 23 Kas 3 20 Kas 2 13 Ns 2 violae (1) 9 Kas 5 mamillatum (1) 7 Ns 2 oligandrum (1) 1 Ss 3 mamillatum (1) Mount Burr forest reserve 21 MBS 2 10 Ys 2 Tantanoola forest reserve <14 Tfs 2 8 Tfs 2 5 Tfs 6 Mount Schank seed orchard 10-11 Bal 13 irregulare (1) vexans (3) Wepar seed orchard 8-9 Kas 9 irregulare (1) mamillatum (4) Mount Gambier forest reserve 39 Csv 7 mastophorum (2) anandrum (2) 39 Ys 6 38 Ys 4 37 Ys 7 mastophorum (1) 21 Ys 3 anandrum (1) 6 MBS 7 2 Ys 3

* Bal, basaltic loam; Csv, coarse sandy valley soil; Kas, Kalangadoo sand; MBS, Mount Burr sand; Ns, Nangwarry sand; Ss, short sand; Tfs, Tantanoola flinty sand; Ys, Young sand. Classification of Stephens et al. (1941). t Number of trees from which the species was isolated. In addition to the Pythium spp. most frequently isolated in the Adelaide hills forest plantations, P. vexans de Bary was isolated from around three trees at Mount Schank seed orchard, and P. violae Chesters & Hickman and P. oligandrum Drechsler 168 E. M. DAVISON AND M. BUMBIERIS were isolated from around one tree each from the Penola forest reserve in the south­ eastern plantations (Table 3). As the association of P. irregulare and P. vexans with decline of woody plants has been shown before (Hendrix et al. 1966) their importance with regard to pine trees needs to be resolved. No correlation could be established between the soil types given in Tables 2 and 3, and the species of fungi isolated from the various sites. Similarly, there was no correlation between the age of sampled trees and the species of fungi isolated from these trees.

V. DISCUSSION Of the Phytophthora spp. isolated from pine plantations in South Australia both P. cinnamomi and P. cryptogea are regarded as serious pathogens of pine trees (Newhook 1959). The isolation of P. cinnamomi from only one tree suggests that this species is not a widely distributed pathogen in South Australian pine plantations. On the other hand, the survey has shown that P. cryptogea is widespread in Kuitpo forest reserve in sites which are subject to waterlogging during wet winters. While there is no direct evidence that the fungus is responsible for death and decline of pine trees in this area, its possible significance with regard to Pinus radiata should be kept in mind when replanting salvaged sites, especially those subject to waterlogging. The fact that the presence of species of Phytophthora and Pythium is not correlated with specific types of symptoms suggest that the sampling technique is recording only part of the population present or some other factor or complex of factors may be involved in the observed disorders of the pine trees.

VI. ACKNOWLEDGMENTS We are grateful to Dr, J. H. Warcup for helpful discussions during this work, and to the Conservator and staff of the Woods and Forests Department of South Australia for their cooperation. One of us (E.M.D.) thanks the Department of Biological Sciences, University of Surrey, for their hospitality whilst working on species identification, and the staff of the Commonwealth Mycological Institute, especially Dr. D. J. Stamps, for provision of cultures and advice.

VII. REFERENCES CHEE, KHENG-Hoy, and NEWHOOK, F. J. (1965).-Improved methods for use in studies on Phytophthora cinnamomi Rands and other Phytophthora species. N.Z. Jl agric. Res. 8, 88-95. COMMONWEALTH OF AUSTRALIA BUREAU OF METEOROLOGY (-).-Meteorological summary. Results of rainfall observations made in South Australia and the Northern Territory 1839-1950. DAVISON, E. M. (1972).-Phytophthora cinnamomi on ornamentals in South A..ustralia. Pl. Dis. Reptr 56, 290. ECKERT, F. M., and TSAO, P. H. (1962).-A selective antibiotic medium for isolation of Phy­ tophthora and Pythium from plant roots. Phytopathology 52,771-7. HENDRIX, F. F. JR., POWELL, W. M., and OWEN, J. H. (1966).-Relation of root necrosis caused by pythium species to peach tree decline. Phytopathology 56, 1229-32. MciNTOSH, D. L. (1964).-Phytophthora spp. in soils of the Okanagan and Simikameen valleys of British Columbia. Can. J. Bot. 42, 1411-15. ----_.-

PHYTOPHTHORA AND PYTHIUM SPP. FROM PINE PLANTATIONS 169

MIDDLETON, J. T. (1943).-The taxonomy, host range and geographic distribution of the genus Pythium. Mem. Torrey bot. Club 20, 1-171. NEWHOOK, F. J. (1959).-The association of Phytophthora spp. with mortality of Pinus radiata and other conifers. I. Symptoms and epidemiology in shelter belts. N.Z. Jl agric. Res. 2, 808-43. OXENHAM, B. L., and WINKS, B. L. (1963).-Phytophthora root rot of Pinus in Queensland. Qd J. agric. Sci. 20, 355-66. PODGER, F. D., and ASHTON, D. H. (1970).-Phytophthora cinnamomi in dying vegetation in the Brisbane ranges, Victoria. Aust. For. Res. 4 (3), 33-6. PODGER, F. D., DOEPEL, R. F., and ZENTMYER, G. A. (1965).-Association of Phytophthora cinnamomi with a disease of Eucalyptus marginata forest in Western Australia. Pl. Dis. Reptr 49,943-7. Rrx, C. E., and HUTTON, J. T. (1953).-A soil survey of the hundred of Kuitpo in the of South Australia. Bull. S.A. Land Tax Dep. No.1. STEPHENS, C. G., CROCKER, R. L., BUTLER, B., and SMITH, R. (1941).-A soil land use survey of the hundreds of Riddock, Hindmarsh, Grey, Young and Nangwarry, County Grey, South Australia. Bull. Coun. scient. indo Res. No. 142. V AARTAJA, O. (1967).-Damping-off pathogens in South Australian nurseries. Phytopathology 57,765-8. VAARTAJA, 0., and BUMBIERIS, M. (1964).-Abundance of pythium species in nursery soils in South Australia. Aust. J. bioI. Sci. 17,436-45. WATERHOUSE, G. M. (1963).-Key to the species of Phytophthora de Bary. C.M.I. Mycol. Pap. No. 92. WATERHOUSE, G. M. (1968).-Key to Pythium Pringsheim. C.M.I. Mycol. Pap. No. 109.