D. Hüberli

False-negative isolations or absence of lesions may cause mis-diagnosis of diseased plants infected with Phytophthora cinnamomi

In a series of growth cabinet, glasshouse and field experiments, tissue samples from living clonal Eucalyptus marginata (jarřah) were incubated immediately after sampling on agar (NARPH) selective for Phytophthora. Phytophthora cinnamomi was recovered 3–6 months after inoculation from 50% of samples with lesions and 30% of symptomless samples. However, up to 11% of samples with and without lesions and from which P. cinnamomi was not initially isolated contained viable pathogen. This was shown by removing tissue which had not produced any growth of P. cinnamomi on NARPH plates, cutting it into smaller sections, washing in sterile deionised water repeatedly for 9 days, and replating. Plating stem or bark tissue directly onto NARPH produced false-negative results for nine P. cinnarnomi isolates and six jarrah clones. The behaviour of the pathogen indicates that it could be present as dormant structures, such as chlamydospores, that need to be induced to germinate. Alternatively, fungistatic compounds in the tissue needed to be removed to allow the pathogen to grow. These results have important implications for disease diagnosis and management, disease-free certification and quarantine clearance.

Fungal Planet description sheets: 107–127

Novel species of microfungi described in the present study include the following from Australia: Phytophthora amnicola from still water, Gnomoniopsis smithogilvyi from Castanea sp., Pseudoplagiostoma corymbiae from Corymbia sp., Diaporthe eucalyptorum from Eucalyptus sp., Sporisorium andrewmitchellii from Enneapogon aff. lindleyanus, Myrmecridium banksiae from Banksia, and Pilidiella wangiensis from Eucalyptus sp. Several species are also described from South Africa, namely: Gondwanamyces wingfieldii from Protea caffra, Montagnula aloes from Aloe sp., Diaporthe canthii from Canthium inerne, Phyllosticta ericarum from Erica gracilis, Coleophoma proteae from Protea caffra, Toxicocladosporium strelitziae from Strelitzia reginae, and Devriesia agapanthi from Agapanthus africanus. Other species include Phytophthora asparagi from Asparagus officinalis (USA), and Diaporthe passiflorae from Passiflora edulis (South America). Furthermore, novel genera of coelomycetes include Chrysocrypta corymbiae from Corymbia sp. (Australia), Trinosporium guianense, isolated as a contaminant (French Guiana), and Xenosonderhenia syzygii, from Syzygium cordatum (South Africa). Pseudopenidiella piceae from Picea abies (Czech Republic), and Phaeocercospora colophospermi from Colophospermum mopane (South Africa) represent novel genera of hyphomycetes. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Fungal Planet description sheets: 69–91

Novel species of microfungi described in the present study include the following from Australia: Bagadiella victoriae and Bagadiella koalae on Eucalyptus spp., Catenulostroma eucalyptorum on Eucalyptus laevopinea, Cercospora eremochloae on Eremochloa bimaculata, Devriesia queenslandica on Scaevola taccada, Diaporthe musigena on Musa sp., Diaporthe acaciigena on Acacia retinodes, Leptoxyphium kurandae on Eucalyptus sp., Neofusicoccum grevilleae on Grevillea aurea, Phytophthora fluvialis from water in native bushland, Pseudocercospora cyathicola on Cyathea australis, and Teratosphaeria mareebensis on Eucalyptus sp. Other species include Passalora leptophlebiae on Eucalyptus leptophlebia (Brazil), Exophiala tremulae on Populus tremuloides and Dictyosporium stellatum from submerged wood (Canada), Mycosphaerella valgourgensis on Yucca sp. (France), Sclerostagonospora cycadis on Cycas revoluta (Japan), Rachicladosporium pini on Pinus monophylla (Netherlands), Mycosphaerella wachendorfiae on Wachendorfia thyrsifolia and Diaporthe rhusicola on Rhus pendulina (South Africa). Novel genera of hyphomycetes include Noosia banksiae on Banksia aemula (Australia), Utrechtiana cibiessia on Phragmites australis (Netherlands), and Funbolia dimorpha on blackened stem bark of an unidentified tree (USA). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Phenotypic Diversification Is Associated with Host- Induced Transposon Derepression in the Sudden Oak Death Pathogen Phytophthora ramorum

The oomycete pathogen Phytophthora ramorum is responsible for sudden oak death (SOD) in California coastal forests. P. ramorum is a generalist pathogen with over 100 known host species. Three or four closely related genotypes of P. ramorum (from a single lineage) were originally introduced in California forests and the pathogen reproduces clonally. Because of this the genetic diversity of P. ramorum is extremely low in Californian forests. However, P. ramorum shows diverse phenotypic variation in colony morphology, colony senescence, and virulence. In this study, we show that phenotypic variation among isolates is associated with the host species from which the microbe was originally cultured. Microarray global mRNA profiling detected derepression of transposable elements (TEs) and down-regulation of crinkler effector homologs (CRNs) in the majority of isolates originating from coast live oak (Quercus agrifolia), but this expression pattern was not observed in isolates from California bay laurel (Umbellularia californica). In some instances, oak and bay laurel isolates originating from the same geographic location had identical genotypes based on multilocus simples sequence repeat (SSR) marker analysis but had different phenotypes. Expression levels of the two marker genes analyzed by quantitative reverse transcription PCR were correlated with originating host species, but not with multilocus genotypes. Because oak is a nontransmissive dead-end host for P. ramorum, our observations are congruent with an epi-transposon hypothesis; that is, physiological stress is triggered on P. ramorum while colonizing oak stems and disrupts epigenetic silencing of TEs. This then results in TE reactivation and possibly genome diversification without significant epidemiological consequences. We propose the P. ramorum-oak host system in California forests as an ad hoc model for epi-transposon mediated diversification.

Phenotypic variation in a clonal lineage of two Phytophthora cinnamomi populations from Western Australia

Seventy-three isolates of Phytophthora cinnamomi were collected from diseased Eucalyptus marginata (jarrah) and Corymbia calophylla (marri) trees in two forest communities in the southwest of Western Australia. Both populations of P. cinnamomi were examined for phenotypic and genotypic variation. Microsatellite DNA analysis showed that all isolates were of the same clonal lineage. We show, for the first time for P. cinnamomi, that morphological and pathogenic variation between populations of the clonal lineage are very broad and continuous. The phenotypes examined included growth rates and colony morphology on potato dextrose agar at different temperatures, sporangial and gametangial morphology, ability to form lesions in detached jarrah and marri stems, and ability to cause deaths of clonal jarrah plants in a glasshouse trial. Phenotype variation was derived asexually. All phenotypes investigated varied independently from one another. Cluster analysis of 24 morphological and pathogenicity phenotypes identified two main clusters of isolates corresponding to each population. The ability to cause deaths in both populations ranged from killing all plants within 59 d to plants being symptomless 182 d after inoculation.

Fishing for Phytophthora from Western Australia’s waterways: a distribution and diversity survey

During one spring season, 12 Phytophthora species, two Phytophthora hybrids, three Halophytophthora species and three Phytopythium species, were isolated from 48 waterways across Western Australia. The waterways were sampled using nylon mesh bags containing leaf baits of up to six different plant species and were isolated by plating necrotic lesions on these onto Phytophthora-selective agar media. Phytophthora species were isolated from all except one waterway. Of the Phytophthora species isolated, eight are known while the remaining four are undescribed taxa. Six of the Phytophthora species and the two hybrids are from clade 6. The two hybrids and P. inundata were the predominant species recovered. Recoveries from different plant leaf baits varied with the best two baits being Pittosporum undulatum and Banksia attentuata; and from these two combined all Phytophthora species were isolated. There was a marked difference in the Phytophthora diversity in the waterways from different regions. This is the first comprehensive study from Australia to examine the Phytophthora communities in waterways, and advances our understanding of the role of these oomycetes in natural and anthropized ecosystems.

Evidence for the role of synchronicity between host phenology and pathogen activity in the distribution of sudden oak death canker disease

Variations in synchronicity between colonization rate by the pathogen and host phenology may account for unexplained spatial distribution of canker disease. The hypothesis that synchronous pathogenicity and host development are necessary for incidence of sudden oak death disease was tested by correlating seasonal variations in host cambial phenology and response to inoculation with Phytophthora ramorum. Response to infection was estimated by inoculating branch cuttings from coast live oak (Quercus agrifolia) trees at nine dates through a full annual cycle in 2003-2004. Host phenology was estimated from measurements of bud burst and cambial activity in spring 2006. Lesions were largest in the spring soon after the cambium resumed activity. A moderate genetic component to lesion size was detected. Variation among trees in date of largest lesions correlated with variation in timing of bud burst and cambial phenology. The data support the hypothesis that active host cambial tissue is a necessary requisite for successful infection with the pathogen that causes sudden oak death canker disease. Genetic variation in host phenology will buffer coast live oak against epidemics of this disease.

First Report on an Infestation of Phytophthora cinnamomi in Natural Oak Woodlands of California and its Differential Impact on Two Native Oak Species

During an intense survey of natural woodlands around Lake Hodges (33°N, 117°W) in June 2001, symptoms typical of root and collar rot caused by Phytophthora spp. were observed on 27% of 474 coast live oaks (Quercus agrifolia Nee.) and on none of 86 Engelmann oaks (Q. engelmannii Greene), in spite of complete spatial intermixing of the two species. Symptoms on coast live oaks included viscous exudates emerging through intact bark matched by underbark dark lesions with irregular margins. Lesions were delineated by a dark line and present on the root collar or the buttress of symptomatic trees. Crowns of trees with lesions ranged from completely healthy to declining or dead. All symptomatic trees were in proximity of the lake or streams. Phytophthora cinnamomi Rands was isolated from four trees in three distinct sites by plating tissues from lesion margins on PARP selective medium and from four soil samples by using standard pear baiting and plating lesions from pear tissue onto PARP. Identification of the isolates was obtained from microscopic observations and direct sequencing of the internal transcribed spacer region of the rDNA (Genbank Accession Nos. AY302148, MC2 and AY302149, MC3). P. citricola Sawada was also isolated once. Pathogenicity tests were completed to compare the susceptibility of the two species of oaks growing in the Lake Hodges region with P. cinnamomi. Two P. cinnamomi isolates from Lake Hodges (MC2, ATCC MYA-3711; MC3) and one isolate from an avocado orchard in San Diego County (MC6) were used to inoculate separately 10 5-year-old trees each of Q. agrifolia and Q. engelmannii grown in 5-gallon containers. Inoculations were performed in two lath-house experiments during February and September 2002 by placing an 8-mm diameter V8-agar plug from the margin of a P. cinnamomi colony underbark and sealing the wound with Parafilm and grafting wax. Lesion lengths were measured 2 months after inoculation, and the presence of the pathogen confirmed by reisolation on PARP. Mean average, maximum, and minimum temperatures were 14, 19, and 9°C and 21, 24, and 18°C for the February and September inoculations, respectively. The February inoculation resulted in small lesions only on Q. agrifolia (26 ± 15 mm, SD). The September inoculation resulted in 135 ± 68 mm (SD) lesions on Q. agrifolia and 49 ± 35 mm (SD) lesions on Q. engelmannii. Controls did not show any lesions. The length of lesions was significantly different between the two hosts (P < 0.0001) and significant differences were observed among the three isolates (P = 0.0018). Although Q. agrifolia is a known host for P. cinnamomi in California (2,3), to our knowledge, this is the first report of widespread infestation of P. cinnamomi in natural oak woodlands in the western United States. Survey and inoculation results indicated Q. engelmannii to be less susceptible to infection. Inoculation results confirm previous research that cold temperatures are unfavorable to this pathogen and isolates differed in pathogenicity toward Q. agrifolia. Decline of oaks infected by P. cinnamomi was observed only in conjunction with other factors, in particular with the presence of the oak twig girdler, Agrilus angelicus Horn., an insect favored by stress conditions such as drought. Similar effects have been reported for Mediterranean oaks infected by the same pathogen (1). References: (1) C. M. Brasier. Nature 360:539, 1992. (2) P. A. Miller. Western Shade Tree Conf. Proc. 8:39, 1941. (3) S. M. Mircetich et al. Plant Dis. Rep. 61:66, 1977.

Temperature and inoculation method influence disease phenotypes and mortality of Eucalyptus marginata clonal lines inoculated with Phytophthora cinnamomi

Survival of l-year-old plants of three clonal lines of Eucalyptus marginata (jarrah), two ranked as resistant (RR1 and RR2) and one as susceptible (SS1) to Phytophthora cinnamomi, was assessed after pathogen inoculation with either mycelial mats underbark or zoospores on the stem. Plants were grown at 15,20, 25 and 30°C. Method of inoculation did not produce comparable mortalities of the clonal lines, particularly at 25 and 30°C. At these temperatures, all three clonal lines had 100% mortality when inoculated underbark, but when inoculated with zoospores, RR1 had 60% survival and lines SS1 and RR2 had 100% mortality. Generally, the level of resistance of all clonal lines declined with increasing temperature. RR2 had consistently higher mortality than SS1, and is therefore not considered resistant. Lesion development was also measured in detached stems of RR1 and a susceptible clonal line (SS2) each inoculated underbark with four different P. cinnamomi isolates. Stems were assessed for lesion development at 20, 25 and 30°C for 4 days. For all four isolates, detached stems of RR1 generally had smaller lesions than those of SS2, particularly at 30°C. The increase in lesion length with increasing temperature was greatest for SS2. Detached stems may have potential in screening for jarrah resistant to P. cinnamomi and allow identification of susceptible clonal lines at 30°C.

First Report of Foliar Infection of Starflower by Phytophthora ramorum

In March 2002, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from pacific or western starflower (Trientalis latifolia Hook.), an herbaceous perennial of the Primulaceae family, at Castro Canyon in Big Sur, Monterey County, California. Affected leaves had numerous necrotic lesions >5 mm in diameter surrounded by a yellow halo, and the lesions coalesced with time. Isolates were identified as P. ramorum by the large chlamydospores, caduceus, semipapillate sporangia, and sequences of the internal transcribed spacer (ITS) region of the rDNA (1,2). The same symptoms were observed on starflower in a second location at the Soquel Demonstration Forest, Santa Cruz County. Although P. ramorum was not isolated from symptomatic leaves on the plants in Santa Cruz County, the ITS region of the pathogen was amplified and sequenced using P. ramorum-specific primers. Both sites were mixed forests of coast redwood (Sequoia sempervirens), bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflorus), which are confirmed hosts of P. ramorum. To test for pathogenicity to starflower, asymptomatic plants were carefully excavated from the two forest locations, replanted in 15-cm paper cups in the original forest soil, and the foliage was inoculated with zoospores of P. ramorum isolate Pr-52, an isolate used in previous inoculations. The zoospores were produced by placing agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar into 20 to 30 ml of sterile deionized water in petri dishes. After 2 days incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then to room temperature for 45 to 60 min. Three hundred μl of the zoospore suspension (approximately 2 × 104 zoospores/ml) was poured into 500-μl modified microcentrifuge tubes in which tips of leaves of starflower were submerged. Control leaves were dipped in sterile deionized water. Plants were placed in a humid-chamber consisting of moist paper towels placed on the tray and covered with a clear-plastic lid that was sprayed with sterile water. The chambers were maintained at 20 to 24°C in the laboratory. Two or three leaves were inoculated, and one leaf was left as the control on each of seven or eight plants in two separate trials. In both trials, water-soaked lesions were observed on the leaves 12 h after inoculation with P. ramorum. At 8 or 11 days after inoculation, necrotic lesions were present on all inoculated leaves starting from the leaf tips. Lesions averaged 29 mm (range 13 to 39 mm) and 45 mm (range 31 to 56 mm) in length in the respective trials. Some lesions covered entire leaves. P. ramorum was reisolated on Phytophthora-selective agar medium (1) from the lesions in both trials. Control leaves had no lesions, and P. ramorum was not reisolated. Infection of starflower and other understory species appears to occur under infested tree hosts such as bay laurel, which is known as a source of inoculum for P. ramorum. To our knowledge, this is the first report of an herbaceous host for P. ramorum and the first report of the disease on the Primulaceae. Previously, only woody hosts were known. Starflower is unlikely to play a major role in the natural spread of the disease, but the pathogen may be spread via movement of plants through the horticultural industry. Furthermore, Trientalis spp. in Europe where P. ramorum is present may also be potential hosts. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.