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.     

Relationships between health of Quercus robur, occurrence of Phytophthora species and site conditions in southern Sweden

The effect of Phytophthora species, soil chemistry, precipitation and temperature on the vitality of oak was evaluated in 32 oak stands in southern Sweden. In addition, the relationship between the occurrence of Phytophthora species and soil conditions was determined. The results showed that there was a weak association between the presence of P. quercina , the most frequently recovered Phytophthora species in southern Sweden, and the vitality of the oak stands (determined from estimates of crown defoliation of individual trees). The pathogens occurred more frequently in clayey and loamy soils that were less acidic and which had higher base saturation. However, they were found in all but the most acidic soils (pH < 3·5). In stands where Phytophthora species were not present, positive correlations between the average crown defoliation and proportion of damaged trees with average summer precipitation and average annual precipitation were found. There were no significant differences in soil chemistry between healthy and declining stands included in this study, and no significant correlations were found between any soil parameter and crown vitality. Based on the results from these 32 oak stands, it is likely that the decline of oaks in southern Sweden can be attributed to several different site-specific factors, such as infection by P. quercina or unusual weather events, which interact with a number of biotic and abiotic factors, leading to oak decline.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

Management of phytophthora root rot in radiata pine seedlings

Chemical and biological agents were evaluated for their ability to suppress root rot, caused by Phytophthora cactorum , in field-grown radiata pine seedlings in New Zealand. Trials were conducted over two seasons in an area of a forest nursery with a natural infestation of P. cactorum , and a history of root rot. In each season, symptoms of root rot developed during April, one month after root pruning, when seedlings were approximately six months old. In trial one, root rot incidence by mid July 2007 was 9·1% in untreated plots and 8·4% in plots that had been treated with metalaxyl-M/mancozeb (14 kg ha⁻¹) at seedling emergence. Disease incidence was lowest (2·1%) in plots that received seven monthly applications of phosphorous acid (6·5 L ha⁻¹). Other treatments, including seed coating with thiram or Trichoderma spp., and foliar applications of methyl jasmonate, did not control disease. In trial two, effects of treatment timing relative to root pruning were investigated. By late June 2008, three months after root pruning, root rot incidence was 22·2% in the untreated plots. Phosphorous acid was the most effective treatment and almost completely suppressed disease (0·1% incidence) when applied fortnightly from February until May (seven applications). Metalaxyl-M/mancozeb (15 kg ha⁻¹) was not effective (21·4% incidence) when applied five months before root pruning. However, disease incidence was reduced when the chemical was applied one week after root pruning (14·9% incidence) and greater control was achieved (8·2% incidence) when the application rate was increased to 50 kg ha⁻¹.     

Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere

To elucidate the mechanism of clubroot suppression under neutral soil pH, a highly reproducible germination assay system under soil culture conditions was designed based on the hypothesis that germinated spores of Plasmodiophora brassicae could be identified by the absence of a nucleus (i.e. having released a zoospore to infect a root hair of the host plant). Brassica rapa var. perviridis seedlings were inoculated with a spore suspension of P. brassicae at a rate of 2·0 × 10⁶ spores g⁻¹ soil and grown in a growth chamber for 7 days. The spores were recovered from rhizosphere and non-rhizosphere soils and stained with both Fluorescent Brightener 28 (cell-wall-specific) and SYTO 82 orange fluorescent nucleic-acid stain (nucleus-specific stain). Total numbers of spores were counted under UV-excitation, and spores with a nucleus that fluoresced orange under G-excitation were counted. The significant increase in the percentage of spores without a nucleus (germinated spores) in the rhizosphere after 7 days' cultivation and the correlation with root-hair infections validated the assay system. Applications of calcium-rich compost or calcium carbonate to neutralize the soil significantly reduced the percentage of germinated spores in the rhizosphere, as well as the number of root-hair infections. The present study provides direct evidence that the inhibition of spore germination is the primary cause of disease suppression under neutral soil pH.     

Control of Phytophthora root rot of irrigated subterranean clover with potassium phosphonate in Victoria, Australia

Phosphonic acid was more effective in inhibiting the production of sporangia of Phytophthora clandestina in sterile pond water (ED₅₀= 1·4 p.p.m.) than it was in inhibiting the growth of mycelium of the fungus on either corn meal agar (ED₅₀= 13·8p.p.m.) or lima bean agar (ED₅₀= 236 p.p.m.). Experiments under controlled environmental conditions showed that better control of tap root rot of subterranean clover caused by P. clandestina was achieved by application of potassium phosphonate to a pasteurized soil mixture than by a spray on the foliage alone. However, in a soil from a pasture, foliar sprays were more effective than soil treatments. Application of superphosphate at a rate of 250 kg/ha or higher to the soil reduced the effectiveness of sprays. In three experiments in irrigated pastures a spray of potassium phosphonate at 300 or 313 ml/ha, applied to cotyledons of subterranean clover and to soil, reduced severity of Phytophthora root rot and increased the annual production of dry matter of the legume by 1·96 to 5·11 t/ha in comparison with untreated controls.     

Phytophthora root and collar rot of alders in Bavaria: distribution, modes of spread and possible management strategies

A survey of symptoms of phytophthora root and collar rot of common ( Alnus glutinosa ) and grey alder ( A. incana ) in riparian and forest stands in Bavaria was conducted by the Bavarian State Forestry and river authorities. Symptoms were seen in 1041 out of 3247 forest alder stands. The majority of the affected stands (80·9%) were less than 21 years old; 46% of these young stands were growing on nonflooded sites and 92% had been planted. The riparian survey showed that symptoms were widespread along more than 50% of the river systems. Along some rivers the disease incidence exceeded 50%. The 'alder Phytophthora ' was recovered from 166 of 185 riparian and forest alder stands with symptoms. In 58 of the 60 rivers and streams investigated in detail, the source of inoculum was traced back to infested young alder plantations growing on the river banks or on forest sites that drain into the rivers. Once introduced to a river system, the 'alder Phytophthora ' infects alders downstream. Baiting tests showed that the 'alder Phytophthora ' was present in rootstocks of alders from three out of four nurseries which regularly bought in alder plants for re-sale, but not in rootstocks from four nurseries that grew their own alders from seed. In addition, the infected nurseries used water from infested water courses for irrigation. The Bavarian State Ministry for Agriculture and Forestry has developed a code of practice for producing healthy alder plants in forest nurseries. This includes a 3-year fallow period between bare-rooted alder crops because of poor survival of the 'alder Phytophthora ' in soil.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Development of a species-specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.     

Quantification of Cylindrocarpon destructans f. sp. panacis in soils by real-time PCR

Ginseng ( Panax quinquefolius ) is an important cash crop in various regions of North America, but yields are often reduced by various root pathogens. A quantitative real-time PCR (qPCR) assay for Cylindrocarpon destructans f. sp. panacis (CDP), the cause of a root rot and replant disease which discourages successive cropping of ginseng on the same site, was developed to quantify the levels of this pathogen in soils previously cropped with ginseng. DNA was extracted from 5-g samples of soil. In pasteurized soils which were re-infested with varying levels of the pathogen, qPCR estimates of pathogen DNA were significantly correlated with disease severity ( r  = 0·494) and with counts of colony-forming units ( r  = 0·620) obtained with an agar medium. In several naturally infested field soils, qPCR estimates of CDP-DNA concentration were significantly correlated with disease severity ( r  = 0·765) and these concentrations were estimated to range from 0 to 1·48 ng g⁻¹ dried soil. A principal components analysis did not show any strong relationships between soil chemistry factors and the concentration of pathogen DNA. The approach outlined here allows the quantification of current populations of CDP in soil many years after ginseng cultivation and the prediction of disease severity in future crops. The method should be generally applicable to root diseases of many crops.