Rapid diagnosis of pathogenic Phytophthora species in soil by real‐time PCR

Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands.     

Specific hybridization real‐time PCR probes for Phytophthora ramorum detection and diagnosis

Sudden Oak Death, caused by Phytophthora ramorum, poses a serious threat to native American oaks, and is also present in Europe where it has been isolated from numerous European ornamental plant nurseries. Its proven aggressiveness against plants in the Fagaceae and Ericaceae and the damage it has caused in North America have lead to it being assigned quarantine status. The timely and accurate detection of P. ramorum is a critical aid in the study of the epidemiology and biology of this pathogen. As a regulated organism, the availability of a sensitive and reliable assay is essential when attempting to achieve early detection of the pathogen. In this work, new specific hybridization probes for a real‐time PCR amplification method were found to be rapid, robust and labour‐saving, and proved suitable for routine use in a molecular diagnostic laboratory.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.     

Development and evaluation of a real‐time PCR seed lot screening method for Fusarium circinatum,causal agent of pitch canker disease

Fusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi‐selective medium and identifying it using morphological traits. This method is time‐consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real‐time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real‐time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10^?5 ng of F. circinatum DNA per PCR. The culture‐based method detected Fusarium spp. in four of six of the same seed lots. The real‐time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture‐based method requires a minimum of 1–2 weeks. This new real‐time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.     

A TaqMan real‐time PCR assay for the detection of Phytophthora ‘taxon Agathis’ in soil,pathogen of Kauri in New Zealand

Kauri Agathis australis, an iconic tree of New Zealand, is under threat from an introduced disease‐causing pathogen provisionally named Phytophthora ‘taxon Agathis’ (referred to as PTA). This soilborne, Pythiaceous species belongs to the Chromista and causes a collar rot resulting in yellowing of the foliage and thinning of the canopy, which eventually causes death of the infected tree. The management and containment of this pathogen requires rapid and reliable detection in the soil. The current method for soil detection utilizes a soil bioassay involving lupin baits and soil flooding in a process that takes between ten and twenty days. We describe a real‐time PCR assay based on TaqMan chemistry for the specific detection of PTA, which targets the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. This TaqMan real‐time PCR assay could be used with DNA extracted directly from bulk soil samples to enable rapid quantification of PTA within soil. The detection limit was 2 fg of PTA DNA from pure culture, or 20 fg in the presence of DNA extracted from soil. The assay was validated using soil samples taken from a PTA‐infested site and soil spiked with a known concentration of oospores. We conclude that the TaqMan real‐time PCR assay offers a more time‐efficient method for detection of PTA in soil than existing methods.     

Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

The development of a species‐specific test to detect Hymenoschyphus pseudoalbidus in ash tissues

Ash dieback, caused by the pathogen Hymenoscyphus pseudoalbidus, is an emerging lethal disease of Fraxinus excelsior in large parts of Europe. To develop a method for the early detection of H. pseudoalbidus, we designed primers for 46 microsatellites (simple sequence repeats, SSRs) of the pathogen. Seven pairs of primers (SSR38, SSR58, SSR114, SSR198, SSR206, SSR211 and SSR212) were found to bind only to the genome of H. pseudoalbidus, but not to the genome of H. albidus or to 52 different fungal endophytes isolated from F. excelsior and F. angustifolia. Using these seven primer pairs, H. pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non‐discoloured wood. Along one twig, H. pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR. Of 50 analysed commercially available seedlings, six were infected with H. pseudoalbidus. Two SSR loci (SSR198 and SSR211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H. pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.     

A method for routine measurements of total sugar and starch content in woody plant tissues

Several extraction and measurement methods currently employed in the determination of total sugar and starch contents in plant tissues were investigated with the view to streamline the process of total sugar and starch determination. Depending on the type and source of tissue, total sugar and starch contents estimated from samples extracted with 80% hot ethanol were significantly greater than from samples extracted with a methanol:chloroform:water solution. The residual ethanol did not interfere with the sugar and starch determination, rendering the removal of ethanol from samples unnecessary. The use of phenol-sulfuric acid with a phenol concentration of 2% provided a relatively simple and reliable colorimetric method to quantify the total soluble-sugar concentration. Performing parallel sugar assays with and without phenol was more useful for accounting for the interfering effects of other substances present in plant tissue than using chloroform. For starch determination, an enzyme mixture of 1000 U alpha-amylase and 5 U amyloglucosidase digested starch in plant tissue samples more rapidly and completely than previously recommended enzyme doses. Dilute sulfuric acid (0.005 N) was less suitable for starch digestion than enzymatic hydrolysis because the acid also broke down structural carbohydrates, resulting in overestimates of starch content. After the enzymatic digestion of starch, the glucose hydrolyzate obtained was measured with a peroxidase-glucose oxidase/o-dianisidine reagent; absorbance being read at 525 nm after the addition of sulfuric acid. With the help of this series of studies, we developed a refined and shortened method suitable for the rapid measurement of total sugar and starch contents in woody plant tissues.     

Agrobacterium pusense,a new plant tumour‐inducing pathogen isolated from Lawson cypress

Lawson cypress (Chamaecyparis lawsoniana), an important landscape tree, is widely planted in gardens and parks throughout Iran. Crown gall disease on Lawson cypress trees was observed in Sari and Juybar Counties, Mazandaran province, northern Iran, in 2017. Isolation from galls on potato dextrose agar (PDA) containing CaCO₃ yielded bacterial colonies, the predominant types of which were purified and selected for characterization. The isolates were Gram‐negative, oxidase positive, able to grow in 2% NaCl and produced 3‐ketolactose. They hydrolysed esculin, casein and arbutin but not starch, gelatin or Tween 80. Two representative isolates were selected for PCR amplification and sequencing of DNA gyrase subunit B (gyrB) gene. In the phylogenetic tree based on the partial sequence of the gyrB gene, isolates KH1 and KH2 clustered with Agrobacterium pusense. The pathogenicity of all isolates was confirmed by inoculation on Jimsonweed (Datura stramonium) and carrot discs (Daucus carota). Confirmation of the presence of genes involved in pathogenicity was made by performing PCR with the virD2A/virD2C and VCF/VCR primer pairs which resulted in amplification of the expected 224 and 730 bp fragments in all studied isolates, respectively. A. pusense was therefore identified as the causal agent of crown and stem gall of Lawson cypress. This appears to be the first report on the natural occurrence of crown gall disease on Lawson cypress and the first record of a plant disease caused by A. pusense.     

Necrophylactic periderm formation in the roots of western larch and Douglas‐fir trees infected with Armillaria ostoyae. I. The response to abiotic wounding in non‐infected roots

The sequence of events leading to necrophylactic periderm formation was studied throughout the year following the abiotic wounding of the non‐infected roots of 10‐ and 27‐year‐old western larch (Larix occidentalis) and 11‐ and 25‐year‐old Douglas‐fir (Pseudotsuga menziesii) trees that were infected with Armillaria ostoyae. The sequence was the same for both ages and species of trees. Wound repair was more rapid in the summer compared with the spring and autumn. Following cell hypertrophy, a zone of lignified impervious tissue was in the initial stages of formation within 10 days of wounding in the summer and 14 days in the spring or autumn. The new phellogen produced a layer of phellem three to four rows of cells thick after 20 days in the summer or 40 days in the spring. Modified cells abutting the inner boundary of the impervious zone frequently developed thick lignified abaxial walls and thin suberized adaxial walls. A typical exophylactic periderm in healthy root bark tissue of both western larch and Douglas‐fir consisted of stone phellem one to four rows of cells thick and a layer of thin‐walled phellem three to six rows of cells thick in western larch and two to three rows thick in Douglas‐fir, a single row of phellogen cells and one to three rows of phelloderm cells. Mature thin‐walled phellem cells had pigmented contents, red in western larch and light brown in Douglas‐fir. In response to wounding, 27‐year‐old western larch and 25‐year‐old Douglas‐fir developed necrophylactic periderms with annual bands of phellem. The bands included a layer of phellem that was six to 12 and nine to 15 rows of cells thicker than the layer of phellem observed in the respective naturally developed exophylactic periderms. Fifty days following wounding in the summer, stone phellem, one to three rows of cells thick, was observed in the necrophylactic periderm of 10‐year‐old trees. When fully developed, the necrophylactic periderm in 27‐year‐old western larch also had a layer of stone phellem three to five rows of cells thick in each band. Stone phellem development was only sporadic in 25‐year‐old Douglas‐fir. Wounds in the winter showed no signs of activity associated with repair until dormancy broke in the spring.     

A new leaf disease on Populus euphratica in south‐western Iran

During the winter of 2012, a leaf spot disease was observed on Euphrates poplar (Populus euphratica) in the forest areas of Khuzestan province, south‐western Iran, causing significant damage in the Karun's riverside forests. Symptoms consisted of necrotic dark brown, circular to oval, 5‐ to 10‐mm spots on both surfaces of the leaves. A fungus having distinct dictyospores similar to those produced by Alternaria spp. was observed. The morphological characteristics, as well as the phylogenetic analysis of the internal transcribed spacer (ITS1‐5,8S‐ITS2) region, confirmed the identity of the strains belonging to the species Alternaria alternata. Pathogenicity tests were conducted on the alive leaves of P. euphratica on the young branches, as well as on the detached leaves in Petri dishes, through inoculation with spore suspension. Target spot symptoms similar to those observed in naturally infected leaves were developed on the inoculated leaves seven to 10 days after inoculation in both the inoculation procedures. A. alternata was consistently re‐isolated from the spots. Interestingly, similar symptoms were observed 7 days after detached leaf treatment with droplets of 15‐day‐old fungal culture filtrate, suggesting the production of pathotoxic compounds by the fungus. To our knowledge, this is the first report of A. alternata causing leaf spot on Euphrates poplar in Iran.     

A new phytoplasma associated with witches’‐broom on Japanese maple in China

In September 2011, five Japanese maple (Acer palmatum Thunb.) trees with symptoms of witches’‐broom were observed growing near each other at a maple grove in Northwest A&F University, Yangling, Shaanxi Province, China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants under transmission electron microscope (TEM). The presence of phytoplasma was further confirmed by a nested polymerase chain reaction (PCR), which amplified a 1.2‐kb fragment using universal primer pair R16mF2/R16mR1 followed by further amplification using primer pair R16F2n/R16R2. Phylogenetic analysis and gel‐based restriction fragment length polymorphism (RFLP) analysis demonstrated that the Japanese maple witches’‐broom was associated with phytoplasma belonging to subgroup 16SrI‐D. This is the first report of a phytoplasma disease of Japanese maple.     

A multiplex one‐step PCR method for the simultaneous identification of Bursaphelenchus xylophilus,B. mucronatus and B. doui– three species within the xylophilus group

The pine wood nematode, Bursaphelenchus xylophilus, causes severe damage to pines in Eastern Asia. Bursaphelenchus mucronatus and B. doui resemble closely B. xylophilus morphologically, moreover they were found frequently in this area recently. It is necessary to identify the three species precisely and rapidly. In this study, we report the results of a multiplex one‐step polymerase chain reaction (PCR) utilizing five primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex one‐step PCR yielded one fragment of about 1000 bp for all Bursaphelenchus populations tested. Futhermore, B. xylophilus, B. mucronatus and B. doui produced another fragment of about 100, 350 and 600 bp respectively. This approach is simple and reliable to simultaneously identify the above three species within the xylophilus group usually encountered together in a nematode assay.     

Variation of endophytic cork oak‐associated fungal communities in relation to plant health and water stress

The main objective of this study was to obtain more comprehensive knowledge about the effect of water stress on endophytic fungal communities in asymptomatic and declining cork oak trees. Six asymptomatic and six declining cork oak trees were randomly selected in a natural cork oak forest located in Sardinia, Italy. In February 2003, the soil around three asymptomatic and three declining trees was covered with a circular plastic film to reduce rain water supply with the intention to induce water stress. The remaining six trees served as controls. Predawn xylematic water potential (PWP) was used as water status indicator and measured seasonally. Between July 2003 and June 2004, fungal endophytes were isolated every 2–4 months from twigs, branches and woody tissues. Significant differences in PWP between covered and control trees were detected mainly in autumn. Gas exchange was greatest in asymptomatic control plants. All tissues were colonized by endophytic fungi. Nineteen fungal species were isolated from 1620 plant fragments. Biscogniauxia mediterranea was the most frequently isolated fungus. Its isolation frequency was significantly higher in declining covered trees than in control trees (p < 0.05). Presence of this fungus in asymptomatic control trees was significantly higher in winter than in summer. Water stress seems to reduce species diversity of the endophytic mycobiota in cork oak and to promote proliferation of some potentially pathogenic endophytes.     

A Method to detect Forest Decline in Germany--Results of a Colour-infra red Aerphotointerpretation

In Northrhine-Westphalia research into the causes of forestdecline is aided by colour infra-red photoflights. For thispurpose some small areas, and several large regions were coveredby flights since 1983. The analysis reported is confined tospruce (Piceaabies (L.) Karst.) because symptoms of new forestdamage are reasonably well understood. Besides normal damage and vigour assessment, additional informationsuch as the social status of individual trees, crown density,sample plot position in the stand and thinning activities werecollected. The main outcome of the data analysis is representedby graphical illustrations. The results indicate that silviculturaltreatments could not have caused the present appearance of sprucebecause these have not changed rapidly in the last decade and,furthermore, the research shows no correlation between thinningactivities and extent of tree stress. On the contrary, dominantand edge trees indicate that all areas show the well known damagesymptoms which are attributable to extreme exposure to rain,fog and wind. The new forest decline is especially unmistakablein older stands. Old trees seem more at risk than young ones. The colour infra-red technique can only indirectly elicit relationshipsbut the results of photointerpretation show it to be a practicableassessment method.