Root infection by Phytophthora cinnamomi in seedlings of three oak species

The combination of soil infestation with Phytophthora cinnamomi and repetitive flooding was studied on 1-year-old plants of Quercus ilex (holm oak) and Q. suber (cork oak). In a second experiment, using 2-year-old plants of the same species and of red oak ( Q. rubra ), the soil infestation was followed by two drought-rewatering cycles. Oak predawn leaf water potential (PLWP) and stomatal conductance (gs) were monitored during both experiments. Root infection, root loss, wilting and mortality were assessed at the end of the experiments. Q. ilex exhibited the highest susceptibility to P. cinnamomi , and Q. rubra the lowest. Root infections caused by P. cinnamomi were more severe in the flooding than in the drought experiment. The most noticeable effect of the infection on plant water relations was a decrease in stomatal conductance. This occurred at different times after inoculation, varying with species susceptibility and experiment. Inoculation with P. cinnamomi induced a decrease of PLWP in Q. ilex plants, and in some Q. suber plants exhibiting a severe root loss. The results further showed that the relationship between PLWP and gs was modified by infection with P. cinnamomi . The combination of flooding and infection with P. cinnamomi acted synergistically on the water relations of Q. ilex . By contrast, there was no significant increase in disease severity due to the postinoculation water stress imposed on the oaks.     

Susceptibility of Iberian trees to Phytophthora ramorum and P. cinnamomi

The capacity of Phytophthora ramorum to colonize the inner bark of 18 native and two exotic tree species from the Iberian Peninsula was tested. Living logs were wound-inoculated in a growth chamber with three isolates belonging to the EU1 and two to the NA1 clonal lineages of P. ramorum . Most of the Quercus species ranked as highly susceptible in experiments carried out in summer, with mean lesion areas over 100 cm² in Q. pubescens , Q. pyrenaica , Q. faginea and Q. suber and as large as 273 cm² in Q. canariensis , ca . 40 days after inoculation. Quercus ilex ranked as moderately susceptible to P. ramorum , forming lesions up to 133 cm² (average 17·2 cm²). Pinus halepensis and P. pinea were highly susceptible, exhibiting long, narrow lesions; but three other pine species, P. pinaster , P. nigra and P. sylvestris , were resistant to slightly susceptible. No significant difference in aggressiveness was found between the isolates of P. ramorum . In addition, there was evidence of genetic variation in susceptibility within host populations, and of significant seasonal variation in host susceptibility in some Quercus species. The results suggest a high risk of some Iberian oaks to P. ramorum , especially in forest ecosystems in southwestern Spain, where relict populations of Q. canariensis grow amongst susceptible understory species such as Rhododendron ponticum and Viburnum tinus . One isolate of P. cinnamomi used as positive control in all the inoculations was also highly aggressive to Iberian oaks and Eucalyptus dalrympleana .     

Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance

Castanea sativa is susceptible to Phytophthora spp., a serious root pathogen causing ink disease, while C. crenata and C. mollissima show resistance to infection. Interspecific controlled crosses were established for introgression of resistance genes from the resistant species into the susceptible C. sativa, and two mapping populations were created. Phytophthora cinnamomi resistance of each progeny was evaluated by root and excised shoot inoculation tests. The number of days of survival after root inoculation was the best discriminator of resistance to P. cinnamomi while the percentage of shoots with internal lesions was the symptom most associated with survival. The lesion progression rate in the excised shoot inoculation test was strongly and negatively correlated with survival in the root inoculation test. The excised shoot inoculation test appears to be a reliable approach for screening the resistance of chestnut genotypes to P. cinnamomi. Strong genetic correlations were obtained between survival and ink disease symptoms and among symptoms, indicating that common or linked genes might influence resistance to P. cinnamomi. The most resistant genotypes selected from this study will be tested for other commercial variables, such as ease of vegetative propagation and stock–scion compatibility.     

Testing variability in pathogenicity of Phytophthora cinnamomi

Forty eight isolates of Phytophthora cinnamomi from various host plants in France (35 isolates) and in other countries were tested for pathogenicity. Seedlings of chestnut, northern red oak, pine and eucalyptus were infected by soil contamination. Taproots, stems and bark strips of plants of chestnut and different oak species were inoculated with mycelium agar disks. Results of the different experiments were in good agreement. All isolates appeared pathogenic to all the different test species but with variable levels of virulence. Isolates with consistent low or high level of virulence, which could be used as standards in further studies, were identified. Interaction between P. cinnamomi isolates and host plant species was significant in terms of lesion lengths. These interactions could not be related to host from which P. cinnamomi was isolated. Consistent with this, in Quercus rubra, the isolate-provenance interaction was not significant. This feature is encouraging for provenance screening for resistance to P. cinnamomi in this species. The variation in virulence was not related to other isolate characteristics (mating type, electrophoretic type, age).     

Effects of Cellulytic Enzymes on Phytophthora cinnamomi

ABSTRACT Two enzyme systems, cellulase (beta-1,4-glucanase) and laminarinase (beta-1,3-glucanase), were added to soil extracts to simulate (in vitro) lytic components found in mulches suppressive to Phytophthora cinnamomi. Concentration ranges of each enzyme were incubated with Phytophthora cinnamomi mycelium, zoospores, zoospores cysts, and zoospore-infected excised roots to evaluate the roles of each enzyme in potential control of avocado root rot disease. Cellulase significantly retarded the development of zoosporangia and chlamydospores when mycelia were incubated in soil extract containing the enzyme at concentrations greater than 10 units/ml. Zoospore production was also reduced by cellulase but not by laminarinase. Laminarinase had little effect on zoosporangia or chlamydospore formation. At high concentrations, laminarinase was consistently more effective at preventing encystment than cellulase. Chlamydospores preformed in root tips were immune to the lytic effects of all treatments except cellulase at 100 units/ml. Zoospores placed in enzyme solutions and plated on a selective medium survived high cellulase concentrations and formed colonies, but there were fewer surviving zoospores when laminarinase was present at greater than 10 units/ml. Low concentrations of cellulase stimulated infection of excised roots, however, low concentrations of laminarinase prevented infection. Cellulase and laminarinase have different effects on the structures of the Phytophthora cinnamomi life history, however, each enzyme may have a role in reduction of inoculum.     

Population Structure of Phytophthora cinnamomi in South Africa

ABSTRACT Phytophthora cinnamomi isolates collected from 1977 to 1986 and 1991 to 1993 in two regions in South Africa were analyzed using isozymes. A total of 135 isolates was analyzed for 14 enzymes representing 20 putative loci, of which four were polymorphic. This led to the identification of nine different multilocus isozyme genotypes. Both mating types of P. cinnamomi occurred commonly in the Cape region, whereas, predominantly, the A2 mating type occurred in the Mpumalanga region of South Africa. A2 mating type isolates could be resolved into seven multilocus isozyme genotypes, compared with only two multilocus isozyme genotypes for the A1 mating type isolates. Low levels of gene (0.115) and genotypic (2.4%) diversity and a low number of alleles per locus (1.43) were observed for the South African P. cinnamomi population. The genetic distance between the Cape and Mpumalanga P. cinnamomi populations was relatively low (D(m) = 0.165), and no specific pattern in regional distribution of multilocus isozyme genotypes could be observed. The genetic distance between the "old" (isolated between 1977 and 1986) and "new" (isolated between 1991 and 1993) P. cinnamomi populations from the Cape was low (D(m) = 0.164), indicating a stable population over time. Three of the nine multilocus isozyme genotypes were specific to the "old" population, and only one multilocus isozyme genotype was specific to the "new" population. Significant differences in allele frequencies, a high genetic distance (D(m) = 0.581) between the Cape A1 and A2 mating type isolates, significant deviations from Hardy-Weinberg equilibrium, a low overall level of heterozygosity, and a high fixation index (0.71) all indicate that sexual reproduction occurs rarely, if at all, in the South African P. cinnamomi population.     

European oak declines and global warming: a theoretical assessment with special reference to the activity of Phytophthora cinnamomi

Causes of current severe declines of the deciduous oaks Quercus robur and Q. petraea in northern and central Europe and of the evergreen Q. ilex, Q. suber and other Quercus spp. in the Mediterranean area are reviewed. Factors implicated include drought, pollution, winter cold, flooding, and stress-related attacks by insects and fungi. Additional factors in Mediterranean oak declines include changing land-use patterns and root disease caused by the aggressive, exotic oomycete root pathogen Phytophthora cinnamomi. Under conditions of global warming the survival and degree of root disease caused by this fungus seems likely to be enhanced, while the host range of the organism might also be increased. Application of the CLIMEX climate-matching program suggests that with a mean increase in temperatures of 1.5–3°C the fungus might considerably increase its disease activity in its existing locations, and to some extent spread northwards and eastwards. However, it seems unlikely to become significantly active in areas of Europe with colder winters such as parts of Scandinavia, Russia and the central Danube. The predictive value of research on major environmental problems such as oak declines could be enhanced by more highly coordinated European forestry research programmes.     

Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi

Although Phytophthora cinnamomi is heterothallic, there are few instances of successful crossing in laboratory experiments, and analysis of field populations indicates a clonally reproducing population. In the absence of sexual recombination, the ability to monitor mitochondrial haplotypes may provide an additional tool for identification of clonal isolates and analysis of population structure. To determine mitochondrial haplotypes for this species, seven mitochondrial loci spanning a total of 6,961 bp were sequenced for 62 isolates representing a geographically diverse collection of isolates with A1 and A2 mating type. Three of the regions were primarily intergenic regions between trnG and rns, rns and nad3, and nad6 and cox1, while the remaining loci spanned cox2, nad9, rps10, and secY coding regions and some of the flanking spacer regions. In total, 45 mitochondrial haplotypes were identified (75% of the total isolates examined) with differences due to single-nucleotide polymorphisms (SNPs, totaling 152 bp) and length mutations (17 indels >2 bp representing a total of 910 bp in length). SNPs were the predominate mutation in the four coding regions and their flanking intergenic regions, while both SNPs and length mutations were observed in the three primarily intergenic regions. Some of the length mutations in these regions were due to addition or loss of unique sequences while others were due to variable numbers of subrepeats (in the trnG-rns region, there were 3 to 12 copies of a 24-bp subrepeat sequence that differentiated 17 haplotypes). Network analysis of the haplotypes identified eight primary clades, with the most divergent clade representing primarily A1 isolates collected from Papua New Guinea. The isolate grouping in the network corresponded to mating type and previously published isozyme classifications, with three exceptions: a haplotype representing an A1 mating type (H29) was placed well within the A2 mating type haplotype grouping, one haplotype (H26) had isolates with two isozyme classifications, and one isozyme group was represented on separate network clades, suggesting that recombination has occurred in the past. Among the 62 isolates examined, several examples were identified of isolates recovered from different geographic regions having the same mitochondrial haplotype, suggesting movement of isolates via plant material. Analysis of the data set to determine whether fewer loci could be sequenced to classify haplotypes indicated that the trnG-rns and rns-nad6 loci would classify 87% of the haplotypes identified in this study, while additional sequencing of the nad9 or secY loci would further differentiate the remaining six haplotypes. Based on conservation of gene order in Phytophthora spp., the trnG-rns locus should be useful for mitochondrial haplotype classification in other species, as should the cox2, nad9, rps10, and secY loci. However, the rns-nad3 and nad6-cox1 loci span regions that can have a different gene order in some Phytophthora spp.     

Variation in Pathogenicity Among South African Isolates of Phytophthora cinnamomi

Phytophthora cinnamomi isolates from South Africa were evaluated for differences in growth rate in vitro and levels of pathogenicity towards Eucalyptus smithii in the field. Inoculations were conducted in the field in summer and winter in two subsequent years at two locations in South Africa using 59 P. cinnamomi isolates. The isolates differed significantly in growth rate in vitro, as well as in levels of pathogenicity to E. smithii in the field. Growth rate in vitro was significantly influenced by interactions with culture age, geographic origin and genetic background as determined using isozymes. Levels of pathogenicity in the field were influenced by season of inoculation and average minimum temperatures at trial sites. The host from which P. cinnamomi isolates were originally obtained did not significantly affect levels of pathogenicity in the field. Culture age had a significant negative effect on growth rate in vitro and pathogenicity in the field. Significant differences in levels of pathogenicity could be found for different multilocus isozyme genotypes. Geographic origin and mating type of P. cinnamomi isolates had no significant effect on levels of pathogenicity in the field. A positive correlation was found between growth rate in vitro and levels of pathogenicity in the field. Levels of variation for pathogenicity within A1 mating type isolates were significantly lower than for A2 mating type isolates. Results of this study provide valuable information on selection of P. cinnamomi isolates for future resistance/tolerance screening assays of Eucalyptus germplasm in South Africa.     

Involvement of soilborne Phytophthora species in Central European oak decline and the effect of site factors on the disease

A survey was made on the occurrence of soilborne Phytophthora species in 35 oak stands on a range of geologically different sites in Bavaria. The most widespread species were P. quercina , P. cambivora and P. citricola . Seven other Phytophthora species were isolated infrequently. The fine root systems of 106 healthy and 111 declining mature trees of Quercus robur and Q. petraea were intensively investigated. The results indicate that, depending on the site conditions, at least two different complex diseases are referred to under the name 'oak decline'. On sites with a mean soil pH (CaCl₂)  3·5 and sandy-loamy to clayey soil texture Phytophthora spp. were commonly isolated from rhizosphere soil, and highly significant correlations existed between crown transparency and various root parameters. Oaks with P. quercina or other Phytophthora spp. in their rhizosphere had markedly higher levels of fine root damage than oaks without Phytophthora spp., and were subject to a relative risk of severe crown symptoms of 2·1 and 2·8, respectively. In contrast, in stands with sandy to sandy-loamy soils and a mean soil pH  3·9, Phytophthora spp. were not found. In these stands, correlations between crown transparency and various root parameters were either less significant or not significant. It is concluded that Phytophthora species are strongly involved in oak decline on sandy-loamy to clayey sites with a mean soil-pH (CaCl₂)  3·5.     

Three Clonal Lineages of Phytophthora cinnamomi in Australia Revealed by Microsatellites

ABSTRACT The genetic structure of populations of Phytophthora cinnamomi, a pathogen of an enormous variety of woody plants, was investigated using microsatellites. Three intensively sampled disease sites in southwest Australia were analyzed along with a large culture collection of Austra-lian isolates and some isolates from elsewhere in the world. The mutation in the four microsatellite loci analyzed revealed spatial patterns at the disease sites that correlated with the age of the infestation. Only three clonal lineages were identified in Australian populations and these same clonal lineages were present in worldwide populations, where it is suggested that a limited number of clonal lineages have spread in most regions. No evidence for sexual reproduction between these clonal lineages in Australia has been found even though the pathogen has the opportunity. Instead, mitotic recombination is frequent within the clonal lineages. The implications of this are discussed.     

Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees

From 1999 to 2001, a survey on the occurrence of Phytophthora spp. in the rhizosphere soil of healthy and declining oak trees was conducted in 51 oak stands in Turkey. Seven Phytophthora spp. were recovered from six out of the nine oak species sampled: P .  cinnamomi , P .  citricola , P .  cryptogea , P .  gonapodyides , P .  quercina , Phytophthora sp. 1 and Phytophthora sp. 2. The most frequently isolated species, P .  quercina , was very common on slopes susceptible to drought. It occurred in four different climatic zones and on six Quercus spp., suggesting that it is native to oaks. The second most common species, P .  citricola , was separated into three subgroups: type C was recovered only in Anatolia, whereas A and B occurred only in the European part of Turkey. Phytophthora cinnamomi was recovered at one site only, and may not be involved in oak decline in Turkey. The other four species were recovered sporadically. On affected sites there was a significant association between deteriorating crown status and the presence of Phytophthora spp., particularly P .  quercina . The occurrence of Phytophthora species was significantly influenced by soil pH. Stem inoculation tests on oak seedlings revealed that Q .  petraea was the most susceptible species.     

Selection for decreased sensitivity to phosphite in Phytophthora cinnamomi with prolonged use of fungicide

To test the hypothesis that resistance in Phytophthora cinnamomi to control by the fungicide phosphite (phosphonate) would arise in sites with prolonged use of phosphite, 30 P. cinnamomi isolates were collected from a range of sites with different phosphite‐use histories, including phosphite‐treated and untreated avocado orchards, and phosphite‐treated and untreated native vegetation sites. The colonizing ability of these isolates was tested by different inoculation methods against a range of host tissues, treated and untreated with phosphite, including mycelial stem inoculation on clonally propagated Leucadendron sp., mycelial root inoculation of lupin seedlings and zoospore inoculation of Eucalyptus sieberi cotyledons. Isolates from avocado orchards with a long history of phosphite use were, on average, more extensive colonizers of the phosphite‐treated Leucadendron sp., lupin seedling roots and Eucalyptus sieberi cotyledons. These isolates did not colonize untreated plant tissue (Leucadendron sp.) more extensively than isolates from sites with no history of phosphite use and no isolates were resistant to control by phosphite. Analysis of all isolates with microsatellite markers revealed the majority were from a single clonal lineage. Selection for decreased sensitivity to phosphite in planta has taken place within asexual clonal lineages of P. cinnamomi in sites with prolonged use of phosphite.     

Mechanisms of resistance to Phytophthora cinnamomi in clonal, micropropagated Eucalyptus marginata

Plants of the eucalypt. Eucalyptus marginata. selected through a glasshouse screening procedure for resistance or susceptibility to Phytophthora cinnamomi , were established in tissue culture and micropropagated. After inoculation with P. cinnamomi , root lesions in clonal lines selected as resistant (RR) to P. cinnamomi were restricted and became contained within four days after inoculation while lesions in roots of those lines susceptible (SS) to P. cinnamomi continued to extend rapidly. Activity of phenylalanine ammonialyase (PAL) was increased above controls in root segments of the RR lines 48 h after inoculation with P. cinnamomi while activity in unselected seedlings and the SS lines was reduced or unchanged. After inoculation, lignin concentration was increased and reached high levels compared with uninoculated control levels in roots of the two RR lines tested. Constitutive levels of phenolics in roots of the RR lines were up to 94% higher than in seedling roots and levels were further increased after inoculation. Levels of phenolics in the other lines and seedlings were unaltered by inoculation. A line derived from resistant seedlings from a susceptible family (RS) had the highest constitutive levels of lignin, which were further increased after inoculation. Resistance to P. cinnamomi in clonally propagated E. marginata seedlings is based on similar mechanisms to those of field resistant species.     

Response of selected South Australian native plant species to Phytophthora cinnamomi

Thirty‐seven South Australian native plant species from 11 families, including 15 threatened species in the state (of which six are listed as threatened under the federal Environment Protection and Biodiversity Conservation Act 1999) were assessed for response to infection by Phytophthora cinnamomi. Seedlings, 3–6 months old and grown in a greenhouse, were inoculated by placing infested pine wood plugs in the potting mix, maintained in moist conditions and assessed for mortality and disease symptoms for between 3 and 10 months. Thirty species were found to be susceptible, of which nine were highly susceptible, 15 moderately susceptible and six slightly susceptible. Three species were found to be resistant and results for four species were inconclusive. Six of the 15 threatened, rare or locally endangered species tested (Eucalyptus viminalis var. viminalis, Correa aemula, C. calycina, Olearia pannosa ssp. pannosa, Pomaderris halmaturina ssp. halmaturina and Prostanthera eurybioides) were moderately susceptible, while two (Allocasuarina robusta and Pultenaea graveolens) were highly susceptible. Significant populations of at least five of the threatened species susceptible to the disease are located close to confirmed or suspected Phytophthora‐infested areas or growing in areas conducive for P. cinnamomi. An effective management strategy is therefore required to avoid extinction of such species due to infection by the phytophthora dieback pathogen.     

Seasonal changes in susceptibility of Quercus suber to Botryosphaeria stevensii and Phytophthora cinnamomi

Monthly inoculations of both intact plants and excised shoots of Quercus suber with the pathogenic species Botryosphaeria stevensii and Phytophthora cinnamomi were performed to investigate seasonal changes in susceptibility of this forest tree species in relation to environmental parameters and plant water status. Infection symptoms were mainly detected on seedlings inoculated from spring to autumn (April through October) with either pathogen. Mean canker sizes also showed a seasonal pattern, the higher values being recorded in the same period as above. Lesion lengths were significantly ( P  < 0·001) related to environmental minimum temperature. Mean daily minimum temperatures within the range of 5–12°C clearly inhibited lesion development of P. cinnamomi , whereas B. stevensii showed a less pronounced decrease in canker expansion at the same temperature range. In excised shoots of Q. suber inoculated monthly with B. stevensii , a negative linear relationship was found between the studied range of plant relative water content (81–91%) and canker length. In contrast, the lesions caused by P. cinnamomi were not significantly ( P  = 0·32) related to any seasonal change in water content. Some control measures for the diseases caused by both pathogens are discussed on the basis of the seasonal changes in host susceptibility observed in this study.     

Penetration of suberized periderm of a woody host by Phytophthora cinnamomi

The mechanisms by which Phytophthora cinnamomi zoospores infect inundated, above‐ground woody stem tissue are described. Using 4–6‐ and 18‐month‐old jarrah seedlings, the infection courts were identified and the invasion of the stems at sites of zoospore cyst binding were described. Stems were inoculated with a suspension of motile zoospores on the green stem/young periderm region. Light microscopy was used to examine penetration at sites of taxis, and fluorescent microscopy was used to examine penetration sites of seedlings with intact periderm. Two main infection courts were identified on stems: the emerging axillary shoot and the region of stem immediately surrounding an axillary shoot, where the periderm was thin or discontinuous. Invasion also occurred at sites where the developing shoot had not yet emerged but was at the stem surface. At these sites the pathogen also directly invaded through the thin‐walled phellem of the periderm surrounding the shoot. Zoospores of P. cinnamomi were not attracted to stomata on mature leaves or green stems. Penetration of the epidermal cell layer of the axillary bud leaf primordia was inter‐ and intra‐cellular; growth of hyphae in the periderm surrounding the shoot was intercellular; while in collenchyma it was inter‐ and intra‐cellular, being intercellular between polyphenolic‐rich cells. Exposed stem collenchyma was also directly invaded immediately adjacent to the young axillary shoot. Zoospores demonstrated taxis to sites of discontinuous periderm, similar to wounded areas where the outer protective layers of the plant are breached. This study presents the first evidence that P. cinnamomi is capable of intercellular penetration of suberized periderm.