Assessment of Australian native annual/herbaceous perennial plant species as asymptomatic or symptomatic hosts of Phytophthora cinnamomi under controlled conditions

Phytophthora cinnamomi is a necrotrophic pathogen of woody perennials and devastates many biomes worldwide. A controlled perlite–hydroponic system with no other hyphae‐producing organisms as contaminants present allowed rapid assessment of ten annual and herbaceous perennial plant species most of which have a wide distribution within the jarrah (Eucalyptus marginata) forest in Western Australia where this pathogen has been introduced. As some annuals and herbaceous perennials have recently been reported as symptomatic and asymptomatic hosts, laboratory screening of some of the field‐tested annuals and herbaceous perennials and additional species was used to further evaluate their role in the pathogen's disease cycle. Nine of the species challenged with the pathogen were asymptomatic, with none developing root lesions; however, Trachymene pilosa died. The pathogen produced thick‐walled chlamydospores and stromata in the asymptomatic roots. Furthermore, haustoria were observed in the roots, indicating that the pathogen was growing as a biotroph in these hosts.     

Evaluation of Juglans species for resistance to Phytophthora cinnamomi: differences in isolate virulence and response to fosetyl‐Al

Phytophthora is considered as an important pathogen on walnut, and severe losses are reported in European as well as in American walnut stands. Though several Phytophthora spp. are known to attack walnut, P. cinnamomi is considered the most virulent and widespread in southern Europe. Up to now, no walnut species or hybrid is known to have a high resistance level towards P. cinnamomi. Efforts are addressed in finding rootstock material graft compatible with English walnut and resistant/tolerant to P. cinnamomi. The extension of P. cinnamomi lesions on five Juglans species was studied to find out sources of resistance/tolerance to this pathogen. Walnut species clustered into two main groups, J. hindsii, J. nigra, and J. mandshurica were the less susceptible to the colonization of P. cinnamomi, while J. regia and J. sieboldiana were the most susceptible. On this account, J. mandshurica represents the best alternative as rootstock because its employment overcomes the risk of the occurrence of black line disease, it has good level of resistance to Agrobacterium temefaciens and Brenneria nigrifluens, and it is tolerant to Xanthomonas arboricola pv. juglandis. J. mandshurica is also compatible in cross‐pollinations with J. regia and J. nigra. Differences in virulence of P. cinnamomi isolates was assessed and a marked interaction between species and isolate emerged. Treatment with fosetyl‐Al by dipping was mainly efficient in reducing the length P. cinnamomi lesions, and an interaction between species and treatment was evident with the highest efficacy on J. regia and J. sieboldiana.     

Temporal metabolic profiling of the Quercus suber–Phytophthora cinnamomi system by middle‐infrared spectroscopy

The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest ‘hotspots’ for biodiversity in the world. Here, we applied Fourier transform‐infrared (FT‐IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P. cinnamomi mycelium during the early hours of the interaction. As early as 2 h post‐inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock‐inoculated plants. These variations were further extended at 8 hpi. Surprisingly, at 16 hpi, the metabolic changes in inoculated and mock‐inoculated plants were similar, and at 24 hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8 hpi. Principal component analysis of the FT‐IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock‐inoculated plants at 2, 8 and 24 hpi, but not at 16 hpi. FT‐IR spectral analysis from mycelium of P. cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24 h post‐exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24 hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT‐IR technique can be successfully applied to discriminate infected cork oak plants from mock‐inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P. cinnamomi were also altered when exposed to cork oak root exudates.     

Involvement of Phytophthora species in white oak (Quercus alba) decline in southern Ohio

This study was initiated to investigate the possible role of Phytophthora species in white oak decline (Quercus alba) in southern Ohio at Scioto Trail State Forest. Surveys demonstrated the presence of four species of Phytophthora including one novel species. By far, the most common species was P. cinnamomi; P. citricola and P. cambivora were isolated infrequently. In few instances, P. cinnamomi was isolated from fine roots and necroses on larger roots. No special pattern of incidence was found, but P. cinnamomi was more commonly isolated from greater Integrated Moisture Index values suggesting moist lower bottomlands favour this Phytophthora species. When tree crown condition was examined relative to the presence of Phytophthora, no significant association was found. However, roots of declining P. cinnamomi‐infested trees had 2.5 times less fine roots than non‐infested and healthy trees, which was significantly different. The population densities of P. cinnamomi from declining trees were significantly greater than from healthy trees, suggesting increased pathogen activity that has the potential to cause dieback and decline and possibly the cause of a reduced fine root amount found on declining trees.     

The role of yellow lupin (Lupinus luteus) in the decline affecting oak agroforestry ecosystems

Phytophthora cinnamomi is a soilborne pathogen causing root rot in Mediterranean Quercus species growing in ‘dehesa’ rangeland ecosystems. Recently, it has been reported causing wilting and death of Lupinus luteus (yellow lupin), a spontaneous plant in southern Spain rangelands, but also frequently sowed for livestock grazing. In soils artificially infested with P. cinnamomi chlamydospores and planted with different cultivars of yellow lupin, a significant increase in the density of propagules was detected in comparison with the initial levels of inoculum and with the infested but not planted soil (control). In oak‐rangelands in which yellow lupine was planted, isolation and counting of colonies of P. cinnamomi from soil samples have shown the ability of this plant to maintain or even increase the inoculum density and thus facilitate the infection of trees. Results suggested that cultivation of yellow lupin in oak‐rangeland ecosystems should be avoided whether oak trees are affected by root disease caused by P. cinnamomi or not. This leguminous plant can act as an inoculum reservoir or even enhance inoculum soil levels available for oak root infections, exacerbating the oak decline severity in the region.     

Screening brassicaceous plants as biofumigants for management of Phytophthora cinnamomi oak disease

Brassicaceous plants rich in glucosinolates have been used as biofumigants for the management of soilborne pathogens. Efficacy of Brassica plant tissue has mainly been attributed to toxic isothiocyanates released upon the hydrolysis of glucosinolates. Management of Phytophthora cinnamomi, the causal agent of oak root rot in rangeland ecosystems using biofumigation, is promising, but requires further validation. The biofumigation activity of 14 brassicaceous plants was evaluated under experimental conditions. All evaluated plants rich in sinigrin suppressed (100%) the mycelial growth of P. cinnamomi, while plants rich in aromatic or other aliphatic glucosinolates had little or no suppressive effect. Simulating soil amendment in field conditions, the effects on natural soil artificially infested with P. cinnamomi chlamydospores were examined with Brassica juncea, Eruca vesicaria and Lepidium sativum, three species with different glucosinolate profiles. Only B. juncea decreased the viability of chlamydospores significantly in comparison with untreated soil only 1 day after biofumigation, whereas E. vesicaria needed 8 days to reach significance and L. sativum had no effect at all. Despite the decreases in soil inoculum, biofumigation with B. juncea did not prevent the root infections in a highly susceptible host (Lupinus luteus). However, biofumigation with plants rich in sinigrin, such as B. juncea, decreased P. cinnamomi soil inoculum under the experimental minimum threshold for oak disease expression. Although biofumigation should be considered as an effective measure to be incorporated in integrated control of the oak disease, biofumigation by itself would not be effective enough for the substantial suppression of P. cinnamomi inoculum.     

The long‐term survival of Phytophthora cinnamomi in mature Banksia grandis killed by the pathogen

The ability of Phytophthora cinnamomi to survive long dry periods is the key to its persistence in the south‐west of Western Australia. It has been proposed that dead Banksia grandis are a significant long‐term reservoir for P. cinnamomi inoculum. To test this, 36 healthy B. grandis trees were inoculated in April 1999, and the presence of viable propagules in planta was determined between 2 and 34 months after tree death. By 10 months after inoculation, 75% of the trees had died, with the remaining seven trees dying by 22 months. The pathogen was more commonly recovered from bark than from wood, except from those trees that died at 22 months, and more commonly from above‐ground trunks than below‐ground trunks and roots until 8 months after plant death. In trees that died 12 months after inoculation, P. cinnamomi was recovered from 60% of trunk and root core samples at 3 months, declining to 33% at 10 months, 5.5% at 12 months and 0.1% at 34 months after tree death. In trees that died at 22 months, P. cinnamomi was recovered from 87% of trunk and root samples 2 months after tree death, decreasing to 0.5% by 33 months. This study suggests that the pathogen does not have a saprotrophic phase within dead B. grandis tissue, and B. grandis is unlikely to be a long‐term reservoir for P. cinnamomi. However, the manipulation of the density of B. grandis and the use of fire to facilitate the breakdown of dead Banksia trunks in the Eucalyptus marginata (jarrah) forest may reduce the spread and impact of P. cinnamomi.     

Fine root dynamics of oak saplings in response to Phytophthora cinnamomi infection under different temperatures and durations

The belowground effects of Phytophthora cinnamomi on 1‐year‐old saplings of two common oak species in mid‐Atlantic US forests, white (Quercus alba) and red oak (Q. rubra), were examined after incubation in pathogen‐infested soilless potting mix. Fine root lengths (0–1.5 mm in diameter) of both oak species were quantified after incubation at successive 30‐day intervals up to 300 days, for a total of 10 incubation periods. In addition, colony‐forming units (CFU) of P. cinnamomi were quantified after white oak saplings were incubated in infested soilless potting mix at different temperature/duration combinations that reflect soil conditions present in the mid‐Atlantic United States. Impact of P. cinnamomi on fine root lengths of red and white oak saplings varied considerably over time. Significant periods of fine root loss occurred primarily during spring, when bud break and leaf flush began for both oak species. Red oaks had 17% fine root loss on average, while white oaks appeared more resistant to P. cinnamomi infection with a 2% decrease in fine roots over the course of the experiment. Phytophthora cinnamomi CFU declined significantly with exposure to all incubation temperatures except 8°C. This was in contrast to in vitro experiments, where the optimum temperature for mycelial growth was determined to be 21°C and above. Significant fine root loss caused by P. cinnamomi depended on plant phenology and the oak species tested. Extreme soil temperatures have a significant adverse impact on temporal changes of P. cinnamomi population.     

Pathogenicity of Phytophthora multivora to Eucalyptus gomphocephala and Eucalyptus marginata

Phytophthora multivora is associated with the rhizosphere of declining Eucalyptus gomphocephala, Eucalyptus marginata and Agonis flexuosa. Two pathogenicity experiments were conducted. The first experiment examined the pathogenicity of five P. multivora isolates and one Phytophthora cinnamomi isolate on the root systems of E. gomphocephala and one P. multivora isolate on the root system of E. marginata. In the second experiment, the pathogenicity of P. multivora to E. gomphocephala and E. marginata saplings was measured using under‐bark stem inoculation. In Experiment 1, the P. cinnamomi isolate was more aggressive than all P. multivora isolates causing significant loss of fine roots and plant death. Two P. multivora isolates and the P. cinnamomi isolate caused significant losses of E. gomphocephala fine roots 0–2 mm in diameter and significantly reduced the surface area of roots 0–1 mm in diameter. One P. multivora and the P. cinnamomi isolate significantly reduced the surface area of roots 1–2 mm in diameter. Two of the P. multivora isolates significantly reduced the number of E. gomphocephala root tips. In E. marginata, the length and surface area of roots 0–1 mm in diameter and number of root tips were significantly reduced by P. multivora infestation. Rhizosphere infestation with the P. multivora isolates and P. cinnamomi isolate on E. gomphocephala, and one P. multivora isolate on E. marginata, did not significantly influence the foliar nutrient concentrations. In Experiment 2, under‐bark inoculation with P. multivora caused significant lesion extension in E. gomphocephala and E. marginata saplings, compared to the control. We propose that P. multivora is inciting E. gomphocephala and E. marginata decline by causing fine root loss and subsequently interfering with nutrient cycling throughout the plant. The impact of fine root loss on the physiology of plants in sites infested with P. multivora requires further research.     

Influence of temperature on germination of Quercus ilex in Phytophthora cinnamomi,P. gonapodyides,P. quercina and P. psychrophila infested soils

The influence of temperature on germination of Quercus ilex acorns in Phytophthora infested soils was quantified for the first time. Radicle damage and mortality of Q. ilex seeds germinating at 17, 20, 23 and 26°C in Phytophthora cinnamomi, P. gonapodyides, P. quercina and P. psychrophila infested soils were assessed and related to in vitro mycelium growth of the same isolates of the pathogens. The optimum growth temperatures of isolates of P. cinnamomi, P. gonapodyides, P. quercina and P. psychrophila were 20–23, 23–26, 20–23 and 20°C, respectively. At 17 and 20°C, all four Phytophthora species caused 100% acorn mortality, whereas at 26°C, acorn mortality was 100, 10, 25 and 0% in P. cinnamomi, P. gonapodyides, P. quercina and P. psychrophila infested soils, respectively. At 23°C, P. cinnamomi and P. gonapodyides reduced acorn radicle length more than P. quercina and P. psychrophila, whereas at 26°C, only P. cinnamomi caused further reduction in radicle length. The higher susceptibility of germinating acorns in comparison to seedlings reported in the literature indicates age‐related susceptibility of Q. ilex to Phytophthora. The seedling/pathogen growth ratio was inversely related to the reduction in radicle length at different temperatures ( = 0.84, p < 0.0001), suggesting that rapid germination may allow seedlings to escape from infection. Increasing temperatures had different effects on damage to acorns depending on the pathogen present in the soil, indicating that Phytophthora species × temperature interactions determined Q. ilex germination. The effects of temperature on the impacts of Phytophthora species based on climate change predictions for Mediterranean countries are discussed.     

Phytophthora species associated with dieback of sweet chestnut in Western Turkey

Sweet chestnut (Castanea sativa) is an important tree species in the Marmara and Aegean regions of Turkey as these two regions produce the great majority of edible nuts, especially those used for marron glacé production. Chestnut forests and orchards in these regions showing severe dieback symptoms not associated with chestnut blight were investigated to determine the role of Phytophthora spp. in the decline syndrome. Soil samples were collected from around 108 symptomatic chestnut trees at 29 sites and Phytophthora spp. isolated using soil baiting technique and selective medium. Species isolated were identified by cultural characteristics and ITS sequencing. Phytophthora x cambivora was the dominant species detected in 13 sites, followed by P. cinnamomi (5 sites), P. plurivora (3 sites) and P. cryptogea (1 site). Phytophthora x cambivora was present in both regions, while P. cinnamomi was found only in the Marmara region in coastal areas around Istanbul. When inoculated at the stem bases of 3‐year‐old chestnut saplings, P. cinnamomi produced significantly longer necrotic lesions (7.8–12.0 cm) than P. x cambivora (2.6–6.3 cm) by 12 days after inoculation. Phytophthora plurivora was the least aggressive species causing only small lesions. Phytophthora cryptogea, which represents the first record on chestnut in Turkey, produced intermediate sized lesions in between P. x cambivora and P. plurivora. These results indicate that P. x cambivora and in some areas P. cinnamomi play major roles in the observed dieback of sweet chestnut in western Turkey.     

The influence of time,soil moisture and exogenous factors on the survival potential of oospores and chlamydospores of Phytophthora cinnamomi

The mode of persistence of Phytophthora cinnamomi, a highly aggressive soil‐ and water‐borne pathogen, remains unclear. This study investigated the survival of viable oospores and chlamydospores of P. cinnamomi when present as free propagules in untreated soil, or in soil subject to four exogenous treatments: smoke water, fish emulsion and two fungicides (ridomil and furalaxyl). The exogenous treatments were applied under moist and dry soil conditions. Spore viability was determined by the thiazolyl blue tetrazolium bromide (MTT) staining technique, with a qPCR assay used to compare general patterns of decline. Over 96% of oospores lost viability over a period of 48 weeks irrespective of soil moisture conditions. The mean percentage viability for oospores decreased from 91% at time zero to 72, 35, 20 and 1% after 6, 12, 24 and 48 weeks, respectively. Reduction in viability of chlamydospores was more rapid than oospores, with viability declining from 92% to zero after 12 weeks. There was no significant difference between untreated soil and the exogenous treatments. The RNA‐based qPCR assay indicated a strong presence of viable oospores of P. cinnamomi up to week 12 for moist soil and week 3 for dry soil, but thereafter failed to detect RNA even though viable oospores could be detected by MTT staining. Based on the MTT staining, this study indicated that viability of P. cinnamomi oospores may be entirely lost within 1 year and that of chlamydospores within 3 months for the soil type tested. Therefore, oospores and chlamydospores when existing as free propagules in soil appear unlikely to be involved in the long‐term survival of P. cinnamomi.     

Involvement of Phytophthora spp. in chestnut decline in the Black Sea region of Turkey

Chestnut blight caused by Cryphonectria parasitica is a serious disease of Castanea sativa in the Black Sea region of Turkey. During disease surveys, dieback and decline symptoms were observed on trees without apparent blight and ink disease symptoms. Black necroses, similar to those caused by Phytophthora infections, were noted on some of the chestnut coppices and saplings in one nursery in Ordu and led to an investigation into this disease complex. Only symptomatic plants showing dieback symptoms were investigated. Soil samples together with fine roots were collected from two directions, north and north‐east, approximately 150 cm away from the main stems. Phytophthora spp. were baited with young chestnut leaves. Three Phytophthora spp., P. cambivora, P. cinnamomi and P. plurivora, were identified from 12 soil samples collected from 73 locations, while from the nurseries, only P. cinnamomi was obtained. Phytophthora cinnamomi was the most common species, obtained from seven locations in five provinces and from four nurseries having similar symptoms mentioned above in different locations. Phytophthora cambivora and P. plurivora were less frequently obtained, from three to two stands, respectively. Phytophthora cinnamomi and P. cambivora were the most aggressive species when inoculated at the stem base on 3‐year‐old chestnut saplings, killing six saplings of eight inoculated in 2 months. The three Phytophthora species were first recorded on chestnut in Black sea region of Turkey with the limited samples investigated in a large area about 150 000 ha chestnut forest.     

Field survey,isolation, identification and pathogenicity of Phytophthora species associated with a Mediterranean‐type tree species

Corymbia calophylla (marri), a keystone tree species in the global biodiversity hot spot of southwestern Australia, is suffering decline and mortality associated with a canker disease caused by the endemic fungus Quambalaria coyrecup. Phytophthora species are frequently isolated from the rhizosphere of C. calophylla, and a hypothesis is that Phytophthora root infection is predisposing C. calophylla to this endemic canker pathogen. Field surveys were conducted in both anthropogenically disturbed and undisturbed C. calophylla stands, from where a total of 100 rhizosphere soil samples, from both healthy and cankered trees, were collected. Phytophthora species were isolated from 26% of the samples collected, with Phytophthora incidence significantly higher on disturbed stands than in natural forests (73% and 27%, respectively). Five Phytophthora species were recovered, including P. cinnamomi, P. elongata, P. multivora, P. pseudocryptogea and P. versiformis. Under‐bark inoculations with the Phytophthora isolates caused significant lesion lengths in excised C. calophylla stems. Corymbia calophylla response to pot infestation trials in the glasshouse varied between Phytophthora species and isolates, with isolates of P. cinnamomi and P. multivora causing a significant reduction in seedling root volume and often leading to seedling death. This study demonstrates that root disease caused by Phytophthora species, especially P. cinnamomi and P. multivora, has the ability to adversely affect C. calophylla health. This study leads the way to do a dual inoculation trial with the canker pathogen Q. coyrecup, and different Phytophthora species to investigate if Phytophthora root infection predisposes C. calophylla to this canker disease.     

Susceptibility to Phytophthora cinnamomi of the commonest morphotypes of Holm oak in southern Spain

The four main morphotypes of Holm oak (Quercus ilex subsp. ballota) present in Andalusia (expansa, macrocarpa, microcarpa and rotundifolia) were infected with Phytophthora cinnamomi to determine their susceptibility to the root pathogen. No large differences were found among the four morphotypes in the infection of roots, which always showed a high degree of necrosis. However, the different responses of the foliage to infection separated the four morphotypes of Holm oak into three groups: very susceptible (microcarpa), susceptible (expansa) and moderately susceptible (rotundifolia and macrocarpa). The natural hybrid Q. ilex ballota–Q. faginea exhibited a low level of root and foliar symptoms when infected with P. cinnamomi. Quercus faginea could be considered as a source of resistance to P. cinnamomi in future breeding programmes.     

Susceptibility of Eucalyptus grandis and Acacia mearnsii seedlings to five Phytophthora species common in South African plantations

Eucalyptus grandis and its hybrids, as well as Acacia mearnsii, are important non‐native trees commonly propagated for forestry purposes in South Africa. In this study, we conducted pathogenicity trials to assess the relative importance of five commonly isolated Phytophthora spp. (Phytophthora alticola, P. cinnamomi, P. frigida, P. multivora and P. nicotianae) from the plantation environment on E. grandis and A. mearnsii seedlings. Overall E. grandis was more susceptible to the tested Phytophthora spp. than A. mearnsii. Phytophthora cinnamomi was the only pathogen that had a significant negative effect on both the host tree species, leading to a reduction in root and shoot weight as well as to death in the case of E. grandis. Phytophthora alticola and P. nicotianae exclusively affected E. grandis and A. mearnsii, respectively. This study updated the current knowledge on the pathogenicity of Phytophthora spp. on two important non‐native commercially propagated tree species from South Africa.     

Influence of site factors on the impact of Phytophthora cinnamomi in cork oak stands in Portugal

Although decline of cork (Quercus suber) and holm oak trees (Quercus rotundifolia) has been described in Portugal in the late years of the 19th century, its development has become a motive of high concern during the last two decades. The presence of Phytophthora cinnamomi in cork and holm oak stands was surveyed in four different regions of the country (Trás‐os‐Montes, Alentejo, Ribatejo and Algarve) during 1995–98. Tree decline severity, sudden death and site characteristics were assessed in 56 sites representing varied conditions. The pathogen was isolated from oak roots and rhizosphere samples in 27 of those places. Other plant species from natural vegetation were sampled in three active disease centres. This survey showed that 56% of the surveyed species of shrub flora were infected with P. cinnamomi, which was detected mainly on species belonging to the families Ericaceae, Cistaceae and Leguminosae. Recovery of P. cinnamomi was more frequent in shallow soils (Leptosols and complex Leptosols and Luvisols). These soils are more common in the south (Algarve), where decline has a high impact. Soils with low fertility and low mineral nutrient levels, particularly phosphorus, seemed to favour infection. Site aspect and topographic tree situation were also evaluated. Sites facing south showed higher occurrence of P. cinnamomi, which was also more frequent in slopes and valleys than on hilltops. In Algarve, a relationship could be established between the crown status and the presence of P. cinnamomi in roots and rhizosphere. Different morphotypes of P. cinnamomi could be distinguished in vitro, and their occurrence in the field was correlated with particular site characteristics. Further research needs and management strategies to limit the extension of the disease are discussed.     

Adaptation of herbaceous plant species in the understorey of <Emphasis Type="Italic">Pinus brutia</Emphasis>

Most of Pinus brutia (Ten.) Holmboe forests are grazed, as silvopastoralism is well adapted in the Mediterranean environment. However, little attention has been paid to the demographic dynamic of the understorey vegetation even though it is strongly affected by the absorbed radiation. The purpose of this study was to examine the adaptation of herbaceous plant species under a Pinus brutia canopy, in Northern Greece. Monocultures of four plant groups (annual and perennial grasses, annual and perennial legumes) were sown in experimental areas of 50%, 70% and 100% light intensity. The density of annual and perennial grasses and perennial legumes were decreased by the shade cast by the Pinus brutia canopy. Population density of the annual legumes was little affected by light intensity: the annual grasses Bromus mollis L. and Lolium rigidum Gaudin; the perennial grasses Dactylis glomerata L. var. palestine, Festuca arundinacea and Agropyron cristatum (L.) Gaertn; the perennial legume Medicago sativa L. var. romana; the annual legumes Medicago lupulina L. and all the tested varieties of Trifolium subterraneum adapted well to the 50% light intensity habitat.     

Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCR

The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth⁺ or TaqF1*. In comparison with baiting (0–10% positive samples), nested PCR proved to be a very much more efficient (90–100% positive samples) method for the detection of P. cinnamomi in soil.