Defining Plant Resistance to Phytophthora cinnamomi: A Standardized Approach to Assessment

Phytophthora cinnamomi is a soil‐borne plant pathogen that causes devastating disease in agricultural and natural systems worldwide. While a small number of species survive infection by the pathogen without producing disease symptoms, the nature of resistance, especially under controlled conditions, remains poorly understood. At present, there are no standardized criteria by which resistance or susceptibility to P. cinnamomi can be assessed, and we have used five parameters consisting of plant fresh weight, root growth, lesion length, relative chlorophyll content of leaves and pathogen colonization of roots to analyse responses to the pathogen. The parameters were tested using two plant species, Zea mays and Lupinus angustifolius, through a time course study of the interactions and resistance and susceptibility defined 7 days after inoculation. A scoring system was devised to enable differentiation of these responses. In the resistant interaction with Z. mays, there was no significant difference in fresh weight, root length and relative chlorophyll content in inoculated compared with control plants. Both lesion size and pathogen colonization of root tissues were limited to the site of inoculation. Following inoculation L. angustifolius showed a significant reduction in plant fresh weight and relative leaf chlorophyll content, cessation of root growth and increased lesion lengths and pathogen colonization. We propose that this technique provides a standardized method for plant–P. cinnamomi interactions that could be widely used to differentiate resistant from susceptible species.     

The abundance of ground-dwelling invertebrates in a Victorian forest affected by ‘dieback’(Phytophthora cinnamomi) disease

Abstract Phytophthora cinnamomi (cinnamon fungus) is a pathogenic soil fungus that infects plant communities along the southeastern coast of Australia and the southwestern corner of Western Australia. Infection of native plant communities with P. cinnamomi regularly leads to dramatic changes in both the structural and the floristic characteristics of these communities. This study aimed to assess the effect of P. cinnamomi induced changes in plant community attributes upon the abundance and diversity of invertebrates in an area of open forest in the Brisbane Ranges, Victoria. Pitfall trapping was conducted continuously for 1 week within each season over a 3 year period. Invertebrates were sorted to order level, and abundances were compared between times (season and year) and infection status for taxa with normalized distributions (Coleoptera, Collembola, Dermaptera and ants). Non-parametric comparisons were made for other groups (Aranae, Blattodea, Diptera, Hemiptera, Orthoptera, ant ‘morpho-species’ and unidentified larvae) to assess differences on the basis of infection status. Significant differences on the basis of infection were uncommon and, where identified, elevated abundances were more commonly observed at sites infected by P. cinnamomi. Consistent temporal effects (season and year) were observed in normalized data sets. Abundances both within individual taxa and from pooled counts were generally weakly associated with ground-level habitat features. Overall, the impact of P. cinnamomi on vegetative structure and floristics was not reflected in different abundances of ground-dwelling invertebrates.     

Characterization of vegetation in an Australian open forest community affected by cinnamon fungus (shape Phytophthora cinnamomi): implications for faunal habitat quality

Phytophthora cinnamomi (cinnamon fungus) is a pathogenic soil fungus which infects plant communities such as open forests, woodlands and heathlands in the south-eastern and south-western corners of Australia, leading to devastating effects upon both structural and floristic features of these plant communities. This study undertaken in open forest in the Brisbane Ranges, south-eastern Australia, sought to characterize two study areas using visual classification and several ordination techniques. Additionally, sites were examined for differences in vegetation composition relating to P. cinnamomi infection, and how these may relate to site habitat quality for resident fauna. This characterization had a high degree of conformity (90% to 96%) between the original visual classification and several ordination methods used. Differences in the cover of understorey vegetation including both low and tall shrubs, and the grass-tree Xanthorrhoea australis were recorded between uninfected and post-infected sites, however the cover of litter or bare ground did not infer infection status. There were no consistent significant differences in projective crown cover of Eucalyptus spp. between uninfected and infected sites, nor in the number of hollows suitable for den or nest sites for arboreal mammals and birds. Hence, the impact of P. cinnamomi upon faunal habitat quality at the sites was considered more likely to relate to changes in ground level vegetation structure than to effects of the pathogen on the dominant Eucalyptus species. Based on the small proportion of sites with detectable isolates of the pathogen (25%), and the slow rate of movement of the disease front during this study (1 to 5 m/3 yr), the current state of disease progression appeared relatively quiescent compared to infestations previously documented in the same area a decade or two earlier.     

Role of Proanthocyanidins in Resistance of the Legume Swainsona formosa to Phytophthora cinnamomi

Reduced flower pigmentation in the legume Swainsona formosa is associated with increased susceptibility to Phytophthora cinnamomi and other soil‐borne pathogens. This study aimed to identify the mechanism for these differences in susceptibility. Chemical analyses of stem tissues that had been previously inoculated with P. cinnamomi revealed that neither anthocyanin nor total phenolic content increased with infection. Such results suggested that observed differences in susceptibility, as indicated by flower colour, were related to preformed rather than induced stem chemistry. Acetone extracts from healthy, uninfected stem tissues of a red‐flowered line were highly toxic to the fungus, while extracts from a white‐flowered line were non‐toxic and those from a pink‐flowered line were intermediate in toxicity and this was correlated with the total phenolic and proanthocyanidin concentration of the extracts. Precipitation of proanthocyanidins with bovine serum albumen removed the toxicity of the extracts. It was concluded that differences in the proanthocyanidin content of tissues contributed to the differences in disease susceptibility of plants with different flower colours.     

Characterization of Phytophthora cinnamomi populations from ornamental plants in South Carolina,USA

Abstract

Fifty-one isolates of Phytophthora cinnamomi isolated from ornamental plants in South Carolina, USA, between 1995 and 2000 were characterized by sporangium morphology, mating type, sensitivity to the fungicide mefenoxam, fatty acid methyl ester (FAME) profile analysis, and amplified fragment length polymorphism (AFLP) analysis. Sporangium shapes were predominantly ovoid to ellipsoid, and size averaged 65.5×40.3 μm (length×breadth) with average length/breadth ratio of 1.6. Forty-nine isolates were the A2 mating type with only two A1 isolates found. This is the first report of the A1 mating type of P. cinnamomi in South Carolina. All isolates were sensitive to mefenoxam and EC₅₀ values for all isolates were less than 0.2 μg ml^?1. FAMEs of each isolate were analysed by gas chromatography and revealed five major fatty acids: myristic (14:0), palmitic (16:0), linoleic (18:2ω6c), oleic (18:1ω9c), and eicosapentaenoic (20:5ω3c) acids. These five fatty acids accounted for more than 80% of FAME profiles. Cluster analysis of FAME profiles showed that individual isolates had unique pattern that could be divided into four major clusters. AFLP analysis based on 200 informative loci also separated isolates into four major clusters. A1 isolates were different from all A2 isolates. The percentage of polymorphic loci (10.5%) and Nei's gene diversity (0.0435) were much higher for the two A1 isolates than for any cluster of A2 isolates even though A2 isolates had more isolates within a cluster. A2 isolates exhibited relatively little genetic variation overall, which suggests that these isolates may have come from a common source.     

Outbreak of Phytophthora cinnamomi causing severe decline of avocado trees in southern Turkey

Since the summer 2017, severe decline symptoms have been observed on 10- to 25-year-old avocado trees in almost all commercial orchards planted in the Mediterranean coastal region of Turkey. Young, newly planted trees in infected orchards were also affected by the disease. Affected trees showed wilting, leaf discoloration, defoliation and severe dieback. Some trees were completely desiccated. Although fine roots of symptomatic trees usually were decayed, reddish brown cankers also occurred on taproots and lateral roots of heavily infected trees. The pathogens were isolated from necrotic root and soil samples of symptomatic trees, using selective medium and soil baiting, and were identified based on morphological features and DNA sequences of the internal transcribed spacer (ITS) region. One isolate each of Phytophthora cryptogea and P. palmivora was identified, while all other isolates were P. cinnamomi. In addition, a subcortical fan-shaped mycelium, characteristic of Armillaria spp., was observed in the stem base of a symptomatic tree and identified as Armillaria gallica by DNA sequences of the internal transcribed spacer (ITS) and the translational elongation factor 1-α (EF 1-α) gene regions. Pathogenicity of Phytophthora isolates was tested by stem inoculation on one-year-old avocado seedlings. Two months after inoculation, canker lesions developed on stems of seedlings inoculated by any of the three Phytophthora spp. In contrast, collenchyma callus formed over the wound points on control plants over the same time period. This is the first report of P. cinnamomi, P. cryptogea, P. palmivora and A. gallica causing root rot of avocado trees in Turkey. In addition, P. cryptogea and A. gallica are reported for the first time associated with disease on this host. Due to the severe symptoms and widespread occurrence, P. cinnamomi should be considered a potential threat to avocado cultivation and natural ecosystems of this region of Turkey.     

Distribution of Phytophthora cinnamomi at different spatial scales: When can a negative result be considered positively?

Abstract In October 1999, patches of dead and dying trees were identified in rainforest vegetation throughout the Tully Falls area in north Queensland, Australia. Previous incidents of patch death in the region had been attributed to Phytophthora cinnamomi. The distribution of P. cinnamomi was assessed by testing for its presence in seven sites displaying signs of dieback and seven sites that appeared healthy. Each site was a circular quadrat, 20‐m radius (total area = 1256.6 m²). Within each quadrat, two perpendicular line transects were constructed. A single soil sample (250 g) was taken at the centre point and at 1‐m intervals along each transect. All soil samples were tested for the presence of P. cinnamomi using a combination of lupin baiting, subsequent culturing and microscopic identification. Of the 1134 samples, 783 recorded positive responses. The mean number of positive responses was not significantly greater in patch death sites than in control sites, suggesting that at this scale of resolution the distribution of P. cinnamomi was uniform. However, at spatial scales of 1‐m intervals across transects the distribution of P. cinnamomi was random.     

Spatial model for predicting the presence of cinnamon fungus (Phytophthora cinnamomi) in sclerophyll vegetation communities in south‐eastern Australia

Abstract The pathogen Phytophthora cinnamomi causes extensive ‘dieback’ of Australian native vegetation. This study investigated the distribution of infection in an area of significant sclerophyll vegetation in Australia. It aimed to determine the relationship of infection to site variables and to develop a predictive model of infection. Site variables recorded at 50 study sites included aspect, slope, altitude, proximity to road and road characteristics, soil profile characteristics and vegetation attributes. Soil and plant tissues were assayed for the presence of the pathogen. A geographical information systyem (GIS) was employed to provide accurate estimations of spatial variables and develop a predictive model for the distribution of P. cinnamomi. The pathogen was isolated from 76% of the study sites. Of the 17 site variables initially investigated during the study a logistic regression model identified only two, elevation and sun‐index, as significant in determining the probability of infection. The presence of P. cinnamomi infection was negatively associated with elevation and positively associated with sun‐index. The model predicted that up to 74% of the study area (11 875 ha) had a high probability of being affected by P. cinnamomi. However, the present areas of infection were small, providing an opportunity for management to minimize spread into highly susceptible uninvaded areas.     

Thermal sensitivity of Phytophthora cinnamomi and long-term effectiveness of soil solarisation to control avocado root rot

Root rot caused by the fungus Phytophthora cinnamomi is a major disease of avocados worldwide. Heat sensitivity of a collection of P. cinnamomi isolates was determined by exposing agar discs containing mycelium or mycelium plus chlamydospores at various temperatures for different periods. Long‐term effectiveness of soil solarisation to control Phytophthora root rot was evaluated in two field trials. In the first, soil disinfestation by solarisation was applied in 1990 to a naturally infested plot before planting avocado (Persea americana) and viñatigo (Persea indica) seedlings. In the second trial, established avocado trees were solarised for four consecutive summers (1996–1999). Results for heat sensitivity showed that fungal mycelium was inactivated after 1–2 h at 38°C. However, 1–2 h at 40°C was needed to kill all propagules when chlamydospores were present. Fungal growth inhibition after thermal treatments was related to levels of time and temperature, and detrimental effects occurred as consequence of sublethal thermal doses. Soil solarisation presented long‐term positive effects when applied as a preplanting treatment. Five years after solarisation, disease severity (0–5 scale where 0 = healthy and 5 = dead plant) of avocado and viñatigo planted in solarised soil was 2.03 and 0.71, respectively, compared with 4.65 and 4.84 in controls. Eleven years after solarisation, the percentage of dead plants in solarised soil was 73% for avocado and 43% for viñatigo but 100% in controls. In contrast, an insufficient level of control was observed in established orchards, probably because of the lower temperature reached during solarisation under the shade of tree canopy. In this situation, maximum temperatures at 5‐cm depth were 10–13.7°C lower than under solar‐heated mulch, only exceeding 40°C in 1997.     

Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.     

A Survey of Phytophthora Species on Hainan Island of South China

During the period 1997–2007, a comprehensive study of the occurrence and distribution of Phytophthora species was conducted on Hainan Island of South China. To date, 14 species of Phytophthora have been recovered and their distribution determined. Phytophthora nicotianae ( =P. parasitica ) is the most important species attacking a wide variety of crops, followed by Phytophthora capsici and Phytophthora citrophthora . In contrast to Phytophthora colocasiae attacking taro leaves throughout the entire island, Phytophthora cyperi was found only once on Digitaria ciliaris in Danzhou. It is of interest to note that Phytophthora heveae, Phytophthora katsurae and Phytophthora insolita are commonly found in forest soil/water of protected mountains without causing any plant diseases. Although Phytophthora species are usually terrestrial or found in fresh water, one isolate of Phytophthora resembling closely the asexual isolates of P. insolita in Hainan was obtained from decaying Rhizophora leaves submerged in seawater. An unidentified Phytophthora species producing non-papillate; internally proliferating sporangia was isolated from the soil in which Ceriops tagel and Bruguiera serangula were growing in a salt water shrimp farm.     

Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant–pathogen interactions in gymnosperm forest trees, including kauri.     

Phytophthora cinnamomi in Shanghai and its possible origin

Camellia leaves were most effective for recoveringPhytophthora cinnamomi from soils followed by azalea leaves and cedar needles. A total of 131 isolates ofP. cinnamomi was obtained from soils and roots collected in Shanghai. Among them 125 were A¹ and 6 were A² type. There was little variation in morphological and physiological characteristics among 82 isolates tested. It is suggested that the fungus may have been a recent settler in Shanghai.     

Phytophthora cinnamomi Associated with Root and Crown Rot of Walnut in Turkey

Walnut decline caused by Phytophthora sp. occurred in an orchard in Sakarya province in Turkey. Affected young trees showed poor growth, leaf discolouration, root and crown rot and eventual death. A Phytophthora sp. isolated from necrotic taproots and crown tissues. The causal agent of the disease was identified as Phytophthora cinnamomi by morphological characteristics and comparing sequences of internal transcribed spacer (ITS) region. Upon conducting pathogenicity test, averaging 7.8‐cm‐long canker developed on basal stem within 2 weeks, while no cankers developed in the control plants.     

Immunological Discrimination of Phytophthora cinnamomi from other Phytophthorae Pathogenic on Chestnut

A polyclonal antiserum (A379) against water soluble proteins from Phytophthora cinnamomi mycelium was produced in rabbit. In ELISA, the 1 : 10 000 diluted antiserum revealed only Phytophthora isolates, not allowing a clear‐cut discrimination among congenerous species, in spite of a generally higher reactivity on P. cinnamomi proteins. The antiserum gave positive reactions in Western blot analyses against mycelial proteins from nine species of Phytophthora and Pythium sp. (grown on rich media), but not with Rhizoctonia solani, binucleate Rhizoctonia, Verticillium dahliae, Fusarium oxysporum and Cryphonectria parasitica. All Phytophthora species showed common epitopes on proteins of molecular masses 77, 66, 51 and 48 kDa. However, a species‐specific protein of 55 kDa was immunodecorated only in P. cinnamomi samples, thus allowing univocal identification of this species. When tested against total proteins from the same fungi grown on water, the antibody revealed diagnostic bands of 55 and 51 kDa in P. cinnamomi only. The antiserum is therefore suitable for the specific identification of P. cinnamomi emerging in distilled water from infected tissues of chestnut, blueberry and azalea.     

Coexistent Mediterranean woody species as a driving factor of Phytophthora cinnamomi infectivity and survival

The long-term conservation of Mediterranean mixed oak forests is seriously threatened by the massive mortality of Quercus suber caused by the exotic pathogen Phytophthora cinnamomi. This species frequently grows in mixed forests under natural conditions, but nothing is known about how its level of disease might be altered by the diversity and identity of coexisting neighbours varying in susceptibility to the exotic pathogen. Here we analysed the individual and combined effects of Q. suber and the main coexisting tree species (Quercus canariensis and Olea europaea subsp. europaea var. sylvestris) in mixed forests of southern Spain on the production of infective and survival spores of P. cinnamomi. Through in vitro experiments, it was demonstrated that mixtures of Q. suber and Q. canariensis highly stimulated the production of P. cinnamomi zoospores in comparison with both species in monocultures. Olea europaea did not stimulate zoospore production. Under controlled conditions, the initial and final densities of inoculum in soil planted with monocultures of O. europaea and Q. canariensis did not differ. However, inoculum densities significantly decreased along the experiment in Q. suber mixtures with O. europaea and Q. canariensis. Phytophthora cinnamomi was able to infect and cause root rot symptoms on all tree species, including O. europaea var. sylvestris. We concluded that mixed stands of Q. suber and Q. canariensis are able to stimulate P. cinnamomi infectivity and survival much more than monospecific stands, and consequently under favourable conditions for root disease development, the coexistence of Q. suber and Q. canariensis might exacerbate Mediterranean forests decline. This study also constitutes the first report of O. europaea var. sylvestris as host and inductor of P. cinnamomi sporulation under controlled conditions.     

Current and projected global distribution of Phytophthora cinnamomi,one of the world's worst plant pathogens

Globally, Phytophthora cinnamomi is listed as one of the 100 worst invasive alien species and active management is required to reduce impact and prevent spread in both horticulture and natural ecosystems. Conversely, there are regions thought to be suitable for the pathogen where no disease is observed. We developed a climex model for the global distribution of P. cinnamomi based on the pathogen's response to temperature and moisture and by incorporating extensive empirical evidence on the presence and absence of the pathogen. The climex model captured areas of climatic suitability where P. cinnamomi occurs that is congruent with all available records. The model was validated by the collection of soil samples from asymptomatic vegetation in areas projected to be suitable by the model for which there were few records. DNA was extracted, and the presence or absence of P. cinnamomi was determined by high‐throughput sequencing (HTS). While not detected using traditional isolation methods, HTS detected P. cinnamomi at higher elevations in eastern Australia and central Tasmania as projected by the climex model. Further support for the climex model was obtained using the large data set from south‐west Australia where the proportion of positive records in an area is related to the Ecoclimatic Index value for the same area. We provide for the first time a comprehensive global map of the current P. cinnamomi distribution, an improved climex model of the distribution, and a projection to 2080 of the distribution with predicted climate change. This information provides the basis for more detailed regional‐scale modelling and supports risk assessment for governments to plan management of this important soil‐borne plant pathogen.