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.     

In vitro leaf inoculation studies as an indication of tree foliage susceptibility to Phytophthora ramorum in the UK

Leaves of 11 coniferous and 23 broad-leaved tree species important to UK forestry were tested for their susceptibility to the quarantine pathogen Phytophthora ramorum using a detached leaf assay. Two European and two USA isolates were used. Wounded and unwounded leaves were dipped in zoospore suspensions during summer; conifers were also tested in winter. Successful infection of tissue and amount of necrosis were assessed. Highly susceptible broad-leaved hosts included Aesculus hippocastanum , Fraxinus excelsior , Quercus ilex , Ulmus procera and, to a lesser extent, Castanea sativa , Q. cerris and Q. petraea , together with Umbellularia californica and rhododendrons. Acer pseudoplatanus , Alnus glutinosa , Carpinus betulus , Corylus avellana , Fagus sylvatica , Prunus avium , Q. robur , Q. rubra and Q. suber showed consistently low susceptibility. Conifer species including Abies procera , Picea abies , P. sitchensis , Pseudotsuga menziesii , Sequoia sempervirens and Tsuga heterophylla were also susceptible. Pseudotsuga menziesii and A. procera were severely affected. Pinus contorta , P. nigra var. maritima and P. sylvestris were virtually resistant, while Taxus baccata was only slightly affected. Increased necrosis was apparent on leaves that were wounded prior to inoculation. These results extend the known range of trees that P. ramorum is able to attack and confirm its relative host-nonspecificity.     

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 .     

Potential susceptibility of Australian native plant species to branch dieback and bole canker diseases caused by Phytophthora ramorum

Susceptibility to branch dieback caused by Phytophthora ramorum was tested using a detached branch assay for 66 Australian native plant species sourced from established gardens and arboreta in California. Six of these species were further tested for their susceptibility to bole cankers caused by P. ramorum using a sealed log assay. Isopogon formosus and Eucalyptus denticulata were identified as potentially highly susceptible Australian branch dieback hosts. Thirteen potentially tolerant Australian host species included Banksia attenuata, B. marginata, E. haemastoma, E. regnans, Pittosporum undulatum and Billardiera heterophylla. Eucalyptus regnans was identified as a potentially highly susceptible bole canker host, while E. diversicolor and E. viminalis were considered potentially tolerant species to bole cankers caused by P. ramorum. Phytophthora ramorum was able to infect all 66 species, as confirmed by reisolation. These results extend the known potential host range for P. ramorum, confirm it as a possible threat to Australian plant industries and ecosystems and highlight additional associated hosts that are important in the global horticultural trade, native forests and plantation forestry.     

Genetic diversity,sensitivity to phenylamide fungicides and aggressiveness of Phytophthora ramorum on Camellia,Rhododendron and Viburnum plants in Spain

Phytophthora ramorum has been detected in official plant health surveys on Rhododendron, Viburnum and Camellia in ornamental nurseries in northern Spain since 2003. A collection of 94 isolates of P. ramorum was obtained from 2003 to 2008 from plants with symptoms at different geographical locations. Isolates were identified based on morphology and sequence of the rDNA ITS region. Mating type, genetic variation, sensitivity to phenylamide fungicides and aggressiveness of these isolates were determined. All isolates belonged to the A1 mating type, ruling out the possibility of genetic recombination. Seven microsatellite markers were used to study genetic diversity; three out of the seven microsatellite markers were polymorphic within the Spanish population of P. ramorum. This study confirms that all Spanish isolates of P. ramorum belonged to the EU1 lineage. Twelve intralineage genotypes were detected, five that are unique to Spain (EU1MG38, EU1MG41, EU1MG37, EU1MG39 and EU1MG40) and seven that are also present in at least one other European country (EU1MG1, EU1MG29, EU1MG22, EU1MG13, EU1MG2, EU1MG18 and EU1MG26). Genotypes EU1MG37, EU1MG39 and EU1MG40 were isolated from Rhododendron from one region; EU1MG38 and EU1MG41 were isolated from Camellia from two different regions. Isolates of genotype EU1MG38 were resistant to metalaxyl and mefenoxam. The level of genetic diversity within the Spanish population of P. ramorum is limited and indicates a relatively recent clonal expansion.     

Diagnostic sensitivity and specificity of different methods used by two laboratories for the detection of Phytophthora ramorum on multiple natural hosts

Five detection methods were comparatively tested on putative Phytophthora ramorum field samples from 41 wild plant species. The tested methods included two culture‐based assays, a DAS‐ELISA‐based polyclonal assay, a nested PCR‐based assay, and a TaqMan real‐time PCR assay. Diagnostic values including sensitivity, specificity, positive predictive value and negative predictive value were calculated for each method. The effects of host species, seasonality and host location were analysed and compared between two laboratories. Significant effects of season, host species and laboratory were detected. It is concluded that a combination of either culturing and molecular diagnosis or of two molecular assays is the most promising approach to diagnose this pathogen. Based on the results of this and other studies, diagnosis should occur as much as possible during wet and warm periods favourable to the pathogen, and proficiency tests should be performed to compare results obtained with molecular approaches in different laboratories. Furthermore, length of time lapsed between sample collection and processing strongly affected the diagnostic sensitivity of culture‐based methods, and therefore needs to be taken into account when comparing results from different laboratories.     

Characterizing the variation in aggressiveness and sporulation of the NA1 and EU1 lineages of Phytophthora ramorum in Oregon

Phytophthora ramorum, the cause of sudden oak death, is an invasive pathogen present in parts of coastal California and south-western Oregon forests. The majority of these forest infestations have been caused by the NA1 clonal lineage. In 2015, the EU1 lineage of P. ramorum was isolated from a tanoak (Notholithocarpus densiflorus) tree located in a mixed-conifer forest of Curry County, Oregon. In order to evaluate the threat to Oregon forests of the EU1 lineage relative to the established NA1 lineage, a series of experiments was conducted comparing aggressiveness and sporulation of NA1 and EU1 isolates on logs and seedlings in the growth chamber and forest. There was no significant difference in lesion size on logs inoculated with NA1 and EU1 isolates for any of the tree species tested. Across all seedling experiments differences among isolates within lineage, in terms of both aggressiveness and sporulation, were more commonly observed than differences among lineages. Site to site variation in tanoak sporulation, as measured by rain bucket baiting, appears to be correlated with the number of P. ramorum-positive seedlings detected at each site.     

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real-time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non-target species.     

Sporulation potential,symptom expression and detection of Phytophthora ramorum on larch needles and other foliar hosts

Phytophthora ramorum has caused extensive dieback and mortality of commercially grown Japanese larch (Larix kaempferi) in many parts of the UK, as infected foliage generates spores that then cause bark lesions and girdling cankers on trees. Following inoculation, individual needles of Japanese, European (L. decidua) and hybrid (L. × eurolepis) larch infected with P. ramorum can produce thousands of sporangia. Mean numbers of sporangia ranged from 806 to 1778 per cm² (hybrid larch and Japanese larch, respectively), surpassing mean sporulation levels on foliar hosts previously associated with P. ramorum outbreaks in Britain, namely Rhododendron ponticum, Castanea sativa and Vaccinium myrtillus. Sporulation on larch even exceeded that of California bay laurel (Umbellularia californica), which drives the sudden oak death epidemic in California. Inoculation of foliage selected at different times of year revealed that foliage age significantly affected sporulation levels, but this varied with host species. However, symptom development and sporulation were often not correlated. Symptoms on larch were frequently insignificant or even absent at certain times of year, with sometimes the only evidence of infection being the emergence of sporangia from needles, without any sign of discolouration or necrosis. Plating infected but symptomless needles onto Phytophthora selective medium also often failed to yield the pathogen. Symptomless infection of larch needles apparently occurs, but is only detectable with microscopy. More generally, it is suggested that diagnosis of Phytophthora infection in conifers is often underestimated due to isolation difficulties and delayed symptom expression.     

Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection

Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real-time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number ( n  = 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20-30% for P. ramorum and P. kernoviae . This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.     

Effects of temperature onPhytophthora porri in vitro,in planta,and in soil

Cardinal temperatures for mycelial growth ofPhytophthora porri on corn-meal agar were <5 (minimum), 15–20 (optimum) and just above 25 °C (maximum). The number of infections after zoospore inoculation of young leaf plants was relatively low at supra-optimal temperatures, but was not affected by sub-optimal temperatures. Even at 0 °C plants were infected. The incubation periods needed for symptom formation were 36–57 d at 0 °C, 13–18 d at 5 °C, and 4–11 d at > 11 °C, and were fitted to temperature between 0 and 24 °C with a hyperbolical model (1/p=0.00812^*T+0.0243). Oospore germination, reported for the first time forP. porri, was strongly reduced after 5 h at 45 °C, and totally absent after 5 h at 55 °C. Soil solarization for six weeks during an exceptionally warm period in May–June 1992 in The Netherlands raised the soil temperature at 5 cm depth for 17 h above 45 °C, but did not reduce the initial level of disease in August significantly.     

In vivo control and in vitro antifungal activity of rhamnolipid B,a glycolipid antibiotic,against Phytophthora capsici and Colletotrichum orbiculare

The glycolipid antibiotic rhamnolipid B isolated from Pseudomonas aeruginosa strain B5 was evaluated for in vitro antifungal activity and in vivo control against phytophthora blight and anthracnose under glasshouse conditions. Rhamnolipid B showed antifungal activity against Cercospora kikuchii, Cladosporium cucumerinum, Colletotrichum orbiculare, Cylindrocarpon destructans, Magnaporthe grisea and Phytophthora capsici. Microscopic observation revealed that the high level of antifungal activity (10 µg ml ⁻¹) against P capsici was mainly due to a lytic effect on zoospores. Zoospore lysis began in the presence of 10 µg ml ⁻¹ of rhamnolipid B and most of the zoospores were collapsed at 25 µg ml ⁻¹. Rhamnolipid B showed inhibitory activity against the germination of zoospores and hyphal growth of P capsici at concentrations of 50 µg ml ⁻¹. Spore germination of the anthracnose plant pathogen C orbiculare was also inhibited in the presence of 50 µg ml ⁻¹ of rhamnolipid B, although hyphal growth was not affected at this concentration. In the glasshouse, the efficacy of rhamnolipid B against phytophthora blight was similar to that of metalaxyl on pepper plants when treated just before inoculation with P capsici. Treatment with either at 500 µg ml ⁻¹ completely protected pepper plants from phytophthora blight. Rhamnolipid B also suppressed the development of C orbiculare infection on leaves of cucumber plants. © 2000 Society of Chemical Industry     

Efficacy of oxolinic acid and other bactericides in suppression ofErwinia amylovora in pear orchards in Israel

The efficacy of oxolinic acid (at 200 and 300 μg a.i./l) and of several antibiotic compounds (streptomycin sulfate at 100 μg a.i./l, glycocide B at 700 μg a.i./l, kasugamycin at 80 μg a.i./l and gentamicin sulfate at 30 and 60 μg a.i./l) againstErwinia amylovora, the causal agent of fire blight in pears, was evaluated in 43 orchard experiments in 1997–2000 in Israel. In addition to the above orchard experiments, the efficacy of the bactericides was tested in live experiments with artificial inoculation. Natural fire blight symptoms were observed in 16 of the 43 experiments; in 13 of them, disease intensity and its distribution among the experimental plots provided a basis for data analysis, leading to reliable conclusions concerning the efficacy of the tested bactericides. Oxolinic acid at 300 μg a.i./l was highly effective againstE. amylovora and reduced disease severity significantly in all experiments, as compared with the untreated plots; however, a concentration of 200 μg a.i./l was not effective in some cases. Among the tested antibiotics, only gentamicin sulfate was as effective as oxolinic acid. Results of the artificial inoculation experiments corroborated those obtained in the naturally infected orchards. The pre-infection activity of oxolinic acid was determined on blossom clusters that were sprayed with the bactericide before inoculation. Control efficacy on blossom clusters sprayed 1–4 days before inoculation ranged from 68% to 80%, a level which did not differ significantly from that observed on blossom clusters sprayed on the day of inoculation (80% control). The postinfection activity of oxolinic acid was determined on blossom clusters that were sprayed with the compound after inoculation. Oxolinic acid was as effective when applied 1 or 2 days after inoculation as when it was applied on the day of inoculation; however, application of the bactericide 3 days after inoculation no longer resulted in significant disease suppression. Oxolinic acid has been used commercially in Israel since 1998 with appreciable success. Contribution No. 533-00 from the Agricultural Research Organization     

Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26

The bacterial strain GC-B26, which showed strong antifungal and anti-oomycete activity against some plant pathogens, was isolated from a grassland soil in Korea. Based on morphological, physiological and biochemical characteristics, GC-B26 was identical to Pseudomonas aeruginosa (Schroeter) Migula. The antibiotic G26A, active against Phytophthora capsici Leonian and Colletotrichum orbiculare (Berk & Mont) van Arx, was isolated from the culture filtrates of Ps aeruginosa strain GC-B26 using various chromatographic procedures. The EI mass and UV spectral results indicated that G26A is an analogue of phenazines, having molecular formula C13H8N2O2 (M+, m/z 224.0664). On the basis of NMR spectral data, G26A was confirmed as phenazine-1-carboxylic acid. C orbiculare, P capsici and Pythium ultimum Trow were most sensitive to G26A, with MIC values of approximately 5 microg ml(-1). However, no antimicrobial activity was found against yeasts and bacteria, even at a concentration of over 100 microg ml(-1). Treatment with the antibiotic gave highly significant protective activity against the development of Phytophthora disease on pepper and anthracnose on cucumber plants. The disease control efficacy was only slightly less than that of the commercial fungicides metalaxyl and chlorothalonil.     

新型植物源农药0.5%大黄素甲醚水剂对黄瓜白粉病菌的毒力测定及田间药效

0.5%大黄素甲醚水剂是湖北省农科院植保土肥所和内蒙古清源保生物科技有限公司联合创制的一种新颖的植物源杀菌剂,其化学结构式为1,8-二羟基-3-甲氧基-6-甲基蒽醌。室内孢子萌发抑制试验结果表明,大黄素甲醚对黄瓜白粉病菌孢子萌发的抑制中浓度EC50为0.37μg/mL;田间药效试验结果表明,0.5%大黄素甲醚水剂对黄瓜白粉病菌具有良好的保护和防治效果,有效成分用量18~45 g/hm2的防治效果明显高于15%三唑酮可湿性粉剂135 g/hm2的防效。     

Antagonistic activity of endophytic fungi towards <Emphasis Type="Italic">Diplodia corticola</Emphasis> assessed by in vitro and in planta tests

One isolate each of Trichoderma viride, Epicoccum nigrum, Fusarium tricinctum, Alternaria alternata, Sclerotinia sclerotiorum and Cytospora (teleomorph: Valsa sp.) present in epigeous declining oak tissues was evaluated for its ability to control Diplodia corticola (isolate 79). This fungus is the causal agent of cankers, vascular necrosis and dieback on various oak species. Among the isolates tested, T. viride and F. tricinctum showed maximum in vitro inhibition of mycelial growth of D. corticola (isolate 79). Species were also evaluated for their ability to reduce mortality caused by D. corticola (isolate 79) of Quercus cerris and Q. pubescens seedlings under controlled conditions. Two series of inoculations were carried out through wounds in the stem; in the first, the distance between the point of inoculation of the antagonist and the pathogen was 6 cm, whereas in the second series the distance was shortened to 3 cm. In seedlings of Q. cerris and Q. pubescens at a distance of 3 cm, inoculation with F. tricinctum and A. alternata significantly reduced mortality caused by D. corticola (isolate 79). Inoculation of T. viride through artificial cuticular wounds in the stem of seedlings prevented the proliferation of D. corticola (isolate 79) only on seedlings of Q. cerris. All Q. pubescens seedlings treated with T. viride manifested pathological symptoms subsequent to proliferation of D. corticola (isolate 79). These observations indicate that the interactions between endophytes in planta and D. corticola (isolate 79) are complex and merit further study.     

Phenotype and spectrum of action of the Pvr4 resistance in pepper against potyviruses, and selection for virulent variants

The dominant Pvr4 gene identified in Capsicum annuum cv. Criollo de Morelos 334 (CM334) is frequently used in pepper cultivars because it possesses one of the largest spectra of action among plant virus resistance genes. This gene was previously shown to confer efficient resistance to all known Potato virus Y isolates, to Pepper mottle virus , to Pepper yellow mosaic virus and to Ecuadorian rocoto virus. This study showed that the W4 line, derived from CM334 and carrying Pvr4 , was also resistant to Peru tomato mosaic virus and Pepper severe mosaic virus , but not to Pepper veinal mottle virus , Chilli veinal mottle virus or Tobacco etch virus . It was noticed that the phenotype of the resistance was atypical since, in the W4 line, hypersensitive reaction or extreme resistance could be observed, depending on virus isolates and inoculated organs. Despite the large deployment of Pvr4 in hybrid cultivars, the numerous tests performed in controlled conditions and the use of W4 serial back-inoculations with potyvirus isolates controlled by this line, no virulent variant isolates were obtained. However, it was shown that the use of graft inoculation experiments allow PVY virulent variants to be selected.     

Variability in aggressiveness,cultural characteristics,cercosporin production and fatty acid profile of Cercospora piaropi,a biocontrol agent of water hyacinth

The extent of variation in aggressiveness, growth and pigmentation in culture, phytotoxin production and fatty acid profile were determined in a population of 55 isolates of Cercospora piaropi, a fungus used as a biocontrol agent of the aquatic weed water hyacinth (Eichhornia crassipes). Besides differences in the colour of mycelium and diffusible pigments in culture, isolates of C. piaropi grown under standard conditions differed significantly in their ability to produce the phytotoxin cercosporin, as well as in aggressiveness and growth rate. A positive correlation existed between the ability of the isolates to produce cercosporin and their aggressiveness, and a negative correlation between growth rate and cercosporin production or growth rate and aggressiveness. Based on thin‐layer chromatographic separation of extracts and comparison with beticolin‐1, used as a standard, there was no evidence of production of beticolins. In discriminant analysis, fatty acid methyl ester (FAME) profiles had low resolution for differentiating populations among isolates of the fungus, and the level of resolution was influenced by the age of the colonies. Diffusible pigments in culture and cercosporin production are useful adjuncts to aggressiveness screening for choosing the most effective isolate of C. piaropi for biological control.