Evaluation of antisera raised against Phytophthora fragariae for detecting the red core disease of strawberries by enzyme-linked immunosorbent assay (ELISA)

Antiserum was raised against pooled mycelial suspensions from five isolates (designated Pf 1, Pf 2, Pf 3, Pf 10 and Pf 11) representing five physiologic races of Phytophthora fragariae. In enzyme-linked immunosorbent assay (ELISA), this antiserum detected homologous soluble antigens at protein concentrations as low as 2 ng/ml.
Fungal antigens could also be detected in extracts of strawberry plants infected with P. fragariae. Root extracts prepared from the alpine strawberry Fragaria vesca and F. ananassa cv. Cambridge Favourite infected with any of the five isolates studied produced strong reactions in ELISA. In F. vesca , ELISA-positive material was detectable 6-8 days after inoculation before macroscopic symptoms appeared. The cultivar Red Gauntlet, which is resistant to Pf 1, 2 and 3 but susceptible to Pf 10 and 11, reflected this differential response in ELISA; the absorbance produced by extracts of plants infected with virulent isolates was significantly higher than that obtained with the corresponding extracts of plants inoculated with a virulent isolates. The recently introduced cultivars Hapil, Ostara and Providence were found to be susceptible to all isolates in this study: the corresponding root extracts were also positive in ELISA.
The antiserum also detected P cactorum infections. Nevertheless, the ELISA test described should prove valuable in screening certified strawberry stocks.     

Presence of Phytophthora fragariae var. fragariae in Norway

The strawberry red core disease caused by Phytophthora fragariae var. fragariae has until recently not been reported from Norway. During 1995 and 1996, samples from 323 Norwegian strawberry fields were tested in a root-tip bait test for possible detection of red core. Symptoms of the disease developed in test plants cultivated in growth medium containing root tips from 21 fields at eight locations in four different counties. Disease symptoms included wilting of plants, root rot, red steles in the roots, and oogonia and oospores identical in size and appearance to those of P.f. fragariae developing along the root steles. In V8 juice agar, the mycelium was slow-growing, thick, fluffy and aerial. Sporangia were large, mainly inversely pyriform, and without papillae. Optimum temperature for mycelial growth was 21°C, and the fungus ceased to grow at 25°C. In infested fields, the most severe attacks were found in low, often poorly drained areas. The means by which red core may have been introduced and spread in the country are discussed, together with preventive action to avoid further dissemination.     

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.     

Survey for Phytophthora fragariae var. fragariae in Finland

A survey for the possible occurrence of red-core disease ( Phytophthora fragariae var. fragariae ) was carried out on 250 strawberry production sites in Finland. The fields were inspected visually. A total of 1080 samples of strawberry roots were taken in spring and autumn 1995 and examined visually, microscopically and by isolation in the laboratory. Root-tip bait-plant tests were performed in the glasshouse to look for the latent presence of the fungus. No red-core disease was detected in any of the inspected fields or in the examined samples.     

Crown rot caused by Phytophthora cactorum in Norwegian strawberry production

Crown rot of strawberry, caused by Phytophthora cactorum , was detected for the first time in Norway in 1992. This paper reports on surveys for P. cactorum in Norwegian certified strawberry plant production and on the distribution of the pathogen in regular strawberry production. In 1996 and 1997, samples of plant material from all certified strawberry plant growers in the country were investigated by isolation on artificial growth medium and using an enzyme-linked immunosorbent assay (ELISA). P. cactorum was not detected in any of the samples. A total of 171 isolations from plants with symptoms resembling crown rot were made from plants in a survey of the distribution of Phytophthora fragariae var. fragariae and from other samples. P. cactorum was detected at 35 different strawberry-producing farms in 11 of the 19 counties of Norway. The fungus was most frequently isolated from cv. Korona (at 18 locations), followed by cv. Inga (at 10 locations).     

Evaluation of root damage to English walnut caused by five Phytophthora species

The pathogenicity of five species of Phytophthora to English walnut was studied in a greenhouse experiment. Phytophthora cinnamomi was the most aggressive species, causing severe root rot and seedling mortality. The other species tested, P. cambivora , P. citricola , P. cactorum and P. cryptogea , did not induce visible crown symptoms on seedlings 2 months after inoculation. Some strains of P. cambivora and P. cactorum also caused taproot damage to seedlings. All except one of the tested isolates caused significant necrosis of fine roots and a significant reduction of root weight compared with noninoculated seedlings. Reduction of above-ground plant development was not statistically significant. While P. cinnamomi is well known as an aggressive primary pathogen of English walnut, the other species of Phytophthora may act as predisposing factors to walnut decline, affecting root system development and increasing host vulnerability to environmental stress.     

Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes

Phytophthora cinnamomi is an ecologically and economically important pathogen. In this study, PCR assays were developed with primer pair LPV2 or LPV3 for rapid detection and identification of this organism. Both primer pairs were selected from putative storage protein genes. The specificity of these primer pairs was evaluated against 49 isolates of P. cinnamomi , 102 isolates from 30 other Phytophthora spp., 17 isolates from nine Pythium spp. and 43 isolates of other water moulds, bacteria and true fungi. PCR with both primer pairs amplified the DNA from all isolates of P. cinnamomi regardless of origin. The LPV3 primers showed adequate specificity among all other species tested. The LPV2 primers cross-reacted with some species of Pythium and true fungi, but not with any other Phytophthora species. PCR with the LPV3 primers detected the pathogen at levels of a single chlamydospore or 10 zoospores in repeated tests. The PCR assay was at least 10 times more sensitive than the plating method for detection of the pathogen from artificially infested soilless medium, and, to a lesser extent, from naturally infected plants. PCR with LPV3 primers can be a useful tool for detecting P. cinnamomi from soilless media and plant tissues at ornamental nurseries, whereas the LPV2 primers can be an effective alternative for identification of this species from pure culture. Applications of these assays for detection of P. cinnamomi in other environments were also discussed.     

Association of Cellulytic Enzyme Activities in Eucalyptus Mulches with Biological Control of Phytophthora cinnamomi

ABSTRACT A series of samples were taken from mulched and unmulched trees starting at the surface of mulch or soil to a 15 cm soil depth, forming a vertical transect. Saprophytic fungi isolated from the soil samples on rose bengal medium and surveyed visually were most abundant in mulches and at the interface of mulch and soil (P < 0.05). Microbial activity as assayed by the hydrolysis of fluorescein diacetate was significantly greater in mulch layers than in soils. Cellulase and laminarinase enzyme activities were greatest in upper mulch layers and rapidly decreased in soil layers (P < 0.05). Enzyme activities against Phytophthora cinnamomi cell walls were significantly greater in mulch than in soil layers. When Phytophthora cinnamomi was incubated in situ at the various transect depths, it was most frequently lysed at the interface between soil and mulch (P < 0.001). Roots that grew in mulch layers were significantly less infected with Phytophthora cinnamomi than roots formed in soil layers. In mulched soil, roots were commonly formed at the mulch-soil interface where Phytophthora populations were reduced, whereas roots in unmulched soil were numerous at the 7.5 cm depth where Phytophthora cinnamomi was prevalent. Enzyme activities were significantly and positively correlated with each other, microbial activity, and saprophytic fungal populations, but significantly and negatively correlated with Phytophthora recovery.     

Novel microsatellite markers for the analysis of Phytophthora infestans populations

Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these, 100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis , P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2–9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa.     

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).     

Survival of Phytophthora ramorum in Rhododendron root balls and in rootless substrates

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium. Following inoculation of the root balls, the aboveground plant parts stayed symptomless, whilst the pathogen could be recovered with a novel non‐destructive baiting assay from the root balls until at least 8 months post‐inoculation. Plating of surface‐sterilized roots and direct microscopic analysis confirmed the presence of P. ramorum in the roots. Phytophthora ramorum could also be baited from the root balls of symptomless Rhododendron plants from commercial nurseries, even 2 years after acquisition. Survival of P. ramorum in rootless media was assessed after burying disks of infected leaf material below the soil surface in columns filled with four different undisturbed forest substrates or a potting medium, and incubated at an outdoor quarantine facility. Phytophthora ramorum could be recovered at least 33 months after burial from all substrates, with a significant increase in recovery after the winter period. These data suggest the possibility for long‐term symptomless presence of P. ramorum in root balls of commercial Rhododendron plants as well as survival in potting medium and different forest substrates under western European climate conditions. Symptomless presence in root balls can contribute to latent spread of this pathogen between nurseries. The novel baiting test, being non‐destructive, simple and applicable to a relatively large number of plants, can offer a valuable tool to test plants for the presence of Phytophthora species in root balls.     

Characterization of Phytophthora spp. Causing Outbreaks of Citrus Brown Rot in Florida

ABSTRACT Epidemics of citrus brown rot from 1994 to 1997 in the south-central and east-coast citrus areas of Florida were characterized and the causal Phytophthora spp. identified. Two species of Phytophthora, P. palmivora and P. nicotianae, were consistently associated with brown rot. Epidemics caused by P. palmivora appeared to be initiated on immature fruit dropped on the orchard floor. The soilborne fungus infected and sporulated on these fruit and was then disseminated to fruit above 1 m in the canopy. In contrast, infection by P. nicotianae, the common cause of root rot, was confined to the lowest 1 m of the canopy. Fruit infected by P. palmivora produced large amounts of ellipsoidal sporangia available for splash dispersal, whereas those infected by P. nicotianae produced far fewer spherical sporangia. Isolates from brown rot epidemics were compared with P. nicotianae from citrus in Florida and Texas, P. citrophthora in California, P. palmivora, and selected Phytophthora spp. from other hosts. Brown rot symptoms produced by the different pathogenic citrus isolates on inoculated fruit were indistinguishable. Morphology, mating behavior, and isozyme patterns of brown rot isolates from 1988 to 1997 matched P. palmivora from citrus roots, other host plants, and other locations, but were different from characterized isolates of P. citrophthora in California and P. nicotianae in Florida and Texas. Cellulose acetate electrophoresis of the isozyme glucose-6-phosphate isomerase rapidly identified the causal citrus pathogen from infected fruit and soil isolation plates. Although P. palmivora is an aggressive pathogen of citrus roots, bark, and fruit, populations in orchard soils were low compared with P. nicotianae.     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

A test system to quantify inoculum in runoff from Phytophthora ramorum-infected plant roots

Foliar hosts of Phytophthora ramorum are often susceptible to root infection but the epidemiological significance of such infections is unknown. A standardized test system was developed to quantify inoculum in runoff from root-infected Viburnum tinus ?Spring Bouquet? or Rhododendron ?Cunningham's White? cuttings. Cuttings of both species gave off a maximum amount of inoculum 1 to 3 weeks after inoculation. The greatest amount of inoculum was recovered from Viburnum roots that were 48 to 70 days old at the time of inoculation, or roots incubated at 15 to 20?C rather than 25?C. Inoculum in runoff from inoculated Viburnum roots was similar for four different isolates of P. ramorum representing both the NA1 and EU1 lineages. When Rhododendron cuttings were inoculated with P. ramorum, P. citricola, or P. cactorum, inoculum of all three pathogens was recovered from runoff, with the highest amount recovered from plants inoculated with P. citricola, followed by the other two. Compared with the other two pathogens, P. ramorum colonized root tissue to a smaller extent. The epidemiology of root infection by P. ramorum is important in itself but the assay might lend itself for use in risk analysis for root infection of other plant species and evaluation of control measures, and also shed light on other root-infecting Phytophthora spp.     

Virulence and aggressiveness of single-zoospore isolates of Phytophthora fragariae

A system for scoring the virulence of isolates of Phytophthora fragariae based on a scale of root rot from 0 ( no symptoms ) to 5 (76-100% roots roiled) on a series of strawberry cultivars is described. Thirty-two single-zoospore isolates from one field site were compared by subjecting their root rot scores to cluster analysis and this grouped them into two major clusters equivalent to physiologic races B66–3 and B66-11, Different sub-clusters of isolates of race B66-11 produced different degrees of rotting on the same hosts. Apart from differences in virulence between the sub-clusters there was some evidence for differences in aggressiveness between isolates within sub-clusters.
Increasing inoculum concentration by over 300-fold increased rotting by c . 25% but did not alter the rankings of different isolate/host combinations. Repeated passage of isolates through cultivars of differing susceptibilities did not affect their pathogenicity.     

利用基因YKT6鉴定疫霉属的不同种

 利用分子标记或对特异位点的碱基序列进行分析是植物病原物分子检测的基础,可以在属和种的水平上对物种进行区分和鉴定。对疫霉属的不同种已有一系列的分子检测方法。SNARE蛋白相关基因YKT6拥有保守的侧翼编码区,适于设计疫霉属特异性的PCR引物,同时其内含子所具有的多态性可开发出几乎所有疫霉种的分子标记。利用疫霉属特异性引物对P-YKT6-F/P-YKT6-R可在31个疫霉种中特异地扩增出一条约600 bp的条带,而在腐霉或其他真菌中不能扩增出该条带。利用大豆疫霉的引物对Ps-YKT6-F/ Ps-YKT6-R和辣椒疫霉的引物对Pc-YKT6-F/Pc-YKT6-R,能分别从大豆疫霉菌株和辣椒疫霉菌株中扩增出一条399 bp和282 bp的条带,常规PCR和巢式PCR的灵敏度分别达到100 pg和10 fg。利用这些引物也可从土壤和病组织中检测到目标病原菌。此外,利用上述特异性引物开发出了大豆疫霉和辣椒疫霉的实时定量PCR检测方法。基于YKT6基因的分子标记和检测方法可用于疫霉种的调查检测和法定定量检测。     

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.     

Estimation of amounts of Phytophthora infestans mycelium in leaf tissue by enzyme-linked immunosorbent assay

A polyclonal antiserum, prepared in a rabbit immunized with a mycelium extract of Phytophthora infestans , reacted in an enzyme-linked immunosorbent assay (ELISA) with mycelial extracts of two Phytophthora species but not with those of 10 other micro-organisms found on potato. P. infestans mycelium in leaf tissue was readily detected by ELISA using either the plate-trapped antigen or F(ab')₂ antibody-fragment techniques. The amount of mycelium in leaf extracts was estimated by comparing the values obtained in ELISA with those for known concentrations of P. infestans mycelium.     

Branch cankers on citrus trees in Spain caused by Phytophthora citrophthora

Considerable losses of citrus trees have been observed in the major citrus-growing areas of Spain. Samples were collected from 132 orchards, and isolations and pathogencity tests were conducted to determine the aetiology of a serious canker disease. Affected trees showed cankers on the scion that frequently began on the branches. Three Phytophthora species were identified based on their morphological, cultural, physiological and molecular profiles. Phytophthora citrophthora was the main species associated with this new syndrome in 114 orchards. Phytophthora nicotianae (syn. P. parasitica ) was isolated from nine orchards as the sole Phytophthora species and in coinfection with P. citrophthora from another nine orchards. Phytophthora citricola was isolated only from one orchard. In stem-inoculation studies conducted under greenhouse conditions, clementine mandarin cv. Hernandina and sweet orange cv. Navel Late were more susceptible to P. citrophthora than sour orange and Carrizo citrange rootstocks. Clementine cv. Hernandina was also highly susceptible in field inoculation experiments. In agreement with field surveys, clementine mandarin cultivars were the most affected, their rootstocks remaining healthy. Phytophthora citrophthora was found to be the predominant species in orchard soils; however, P. nicotianae was also isolated. This information changes the scenario of diseases caused by Phytophthora spp. in Spain and consequently, the present knowledge of epidemiology and the effectiveness of the current control measures should be reassessed.     

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.