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.     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Survival of Phytophthora cinnamomi in Buried Eucalyptus Roots

Colonization and survival of Phytophthora cinnamomi in roots was tested in 3 months old, axenically grown seedlings of Eucalyptus maculata (field resistant) and E. sieberi (susceptible). The roots were inoculated, then one week later were excised and buried in three non-sterile, conducive soils; a lateritic gravel, an infertile duplex soil, a loamy sand as well as in a fertile, suppressive krasnozem. Pathogen viability, percentage root colonization and chlamydospore numbers were examined at matric potentials of ?1/3, ?5 and ?10 bar after periods of 10, 100 and 200 days at 21°C. At 10 days, survival was 100% in the form of mycelium and the only significant difference was between the two Eucalyptus species. At 100 days survival was solely due to chlamydospores, but the pathogen was viable in all inoculated roots and at each matric potential. At 200 days soils had dried to less than ?10 bars and the pathogen failed to survive. No significant differences were found between the two pathogen isolates but significant differences were obtained between the susceptible and field resistant Eucalyptus species. Pathogen viability, percentage root colonization and chlamydospore number were highly correlated with soil types and matric potential. These components declined with decreasing soil matric potential. The Krasnozem was only suppressive at relatively high soil matric potentials (?1/3 bar). At lower values (?5, ?10 bar) survival of the pathogen, chlamydospore numbers and percentage colonization of the roots in the Krasnozem were comparable with that of the 3 conducive soils tested. Chlamydospores were present, but in low numbers in roots buried in the suppressive soil at ?1/3 bar.     

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.     

Comparison of damage to native and exotic tallgrass prairie plants by natural enemies

We surveyed the prevalence and amount of leaf damage related to herbivory and pathogens on 12 pairs of exotic (invasive and noninvasive) and ecologically similar native plant species in tallgrass prairie to examine whether patterns of damage match predictions from the enemy release hypothesis. We also assessed whether natural enemy impacts differed in response to key environmental factors in tallgrass prairie by surveying the prevalence of rust on the dominant C₄ grass, Andropogon gerardii, and its congeneric invasive exotic C₄ grass, A. bladhii, in response to fire and nitrogen fertilization treatments. Overall, we found that the native species sustain 56.4% more overall leaf damage and 83.6% more herbivore-related leaf damage when compared to the exotic species. Moreover, we found that the invasive exotic species sustained less damage from enemies relative to their corresponding native species than the noninvasive exotic species. Finally, we found that burning and nitrogen fertilization both significantly increased the prevalence of rust fungi in the native grass, while rust fungi rarely occurred on the exotic grass. These results indicate that reduced damage from enemies may in part explain the successful naturalization of exotic species and the spread of invasive exotic species in tallgrass prairie.     

Benzoic acid induces tolerance to biotic stress caused by Phytophthora cinnamomi in Banksia attenuata

Banksia attenuata plants were treated with soil drenches or foliar sprays of benzoic acid (BZA) to determine induced resistance to Phytophthora cinnamomi. Stems of B. attenuata were inoculated with the pathogen 1 week after treatment with BZA. Resistance was estimated by measuring P. cinnamomi lesions on stems. Treatment with 0.10 mM, 0.25 mM or 0.50 mM BZA caused a reduction in lesion size with 0.50 mM BZA applied as a soil drench being the most effective treatment at suppressing the development of lesions. This is the first report of BZA induced host resistance in any plant species to any pathogen.     

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.     

Identification and characterization of superoxide dismutase in Phytophthora cinnamomi

Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O₂ ^?), which was added exogenously. Our results suggest that the metabolism of O₂ ^? has an important role in the development of P. cinnamomi.     

Phytophthora cinnamomi: the Dynamics of Chlamydospore Formation and Survival

Chlamydospore activity was investigated by inoculating 10 day old seedlings of Lupinus angustifolius with Phytophthora cinnamomi. Infected roots were excised and buried in 4 non-sterile media: – glass beads, gravel, sand or soil at different water potentials and incubated at 22°C. Roots were examined and chlamydospores counted initially and after 10, 20 and 30 days. Large numbers of spores formed in the roots buried in the various media. Spore formation was rapid in glass beads and gravel, and slower in soil, but more chlamydospores survived the 30 day period when roots were buried in soil. Nylon mesh squares 1 cm²were inoculated with 38 chlamydospores and buried in the same 4 media. 70–100% of chlamydospores germinated and colonised 90–100% of the mesh, producing mycelia and an increased number of chlamydospores. In some instances there was a ten-fold increase in spore numbers and numbers were still increasing at 28 days. Maximum numbers and maximum survival occurred on mesh buried in soil. The role of the chlamydospores is considered, as that of a dynamic unit with a saprobic phase in the life cycle of the fungus which is independent of host, and has the capacity to increase both population density and distribution of the pathogen.     

Changes in Respiration of Seedling Roots Inoculated with Phytophthora cinnamomi

An increase in rate of respiration was recorded for intact roots of seven native Australian species 16 h after inoculation with Phytophthora cinnamomi. By 24 h the magnitude of the increase ranged from 2—159% above that of the uninoculated controls and was evidently not related to host susceptibility. A time sequence study of lesion extension and the associated increased respiration rates for both susceptible and tolerant eucalypts demonstrated a difference in response. The rate of respiration in the tolerant species increased 2 % and only at the site of inoculation, whereas in the susceptible species the respiration rate increased in a wave which began at the inoculation site and continued along the root with the advancing fungal invasion. Respiration rate only increased in regions of the root actually inhabited by the pathogen. The fungal contribution to the total respiration of infected roots was less than 1 % and was determined by measuring respiration of inoculated killed roots. Respiration rates were measured in the presence of potassium cyanide (KCN) and salicylhydroxamic acid (SHAM). Both KCN-sensitive and SHAM-sensitive respiration occurred in normal uninfected E. marginate seedlings. A large proportion of the increase in total respiration rate of infected seedlings compared with uninoculated controls was due to the alternate, SHAM-sensitive pathway. The physiological implications of these results are discussed.     

Genetic variation in resistance to Phytophthora cinnamomi in seedlings of two Turkish Abies species

Genetic variation in resistance to Phytophthora cinnamomi was investigated for two fir species endemic to the Republic of Turkey. Open-pollinated families of seedlings of Trojan fir (Abies equi-trojani) and Turkish fir (Abies bornmuelleriana) were grown from seed in a greenhouse for approximately 15 months, inoculated with rice grains colonized with P. cinnamomi, and subsequent mortality assessed biweekly for 16 weeks. Final seedling mortality was higher in Trojan fir (56.4 %) compared to Turkish fir (32.9 %). Mortality in both species varied by geographic origin, decreasing from west (59.8 %, ) to east (21.4 %, Karabük). As mortality increased following inoculation, both narrow-sense individual-tree $ \left( {h_i^2} \right) $ and family mean $ ( {h_f^2} ) $ heritabilities increased, plateauing at 0.62?±?0.162 and 0.97?±?0.011 for Trojan fir and 0.50?±?0.102 and 0.96?±?0.01 for Turkish fir, respectively. Terminal and lateral branch bud break assessed under greenhouse conditions were also under strong genetic control. For terminal bud break, individual-tree heritabilities for Trojan and Turkish fir were 0.49?±?0.146 and 0.45?±?0.099, respectively, while family mean heritabilities were 0.88?±?0.035 and 0.88?±?0.027, respectively. The family mean correlation between bud break and final disease mortality was not significant for lateral buds but positive and significant for terminal buds (r?=?0.32) suggesting that selection for resistance would either not alter, or slightly reduce, early bud break. These are encouraging results for ongoing tree improvement efforts in North America and Europe to develop planting stock for the Christmas tree industry.