郴州市松材线虫病防治方法评析

对郴州市松材线虫病发生情况进行分析,对采取的几种防治方法进行评析,试图为今后防治松材线虫病提供思路。     

Localized within‐ and between‐tree variation in nematode distribution during latent state of pine wilt disease makes the disease status cryptic

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, an epidemic disease that has severely damaged pine forests in East Asia. The disease has spread to northern areas in Asia and parts of Europe. To prevent disease spread as the forefront of damage prevention, a better understanding of infection status is highly important. Not all infected trees show disease symptoms, and such asymptomatic PWN‐carrying trees are likely to be overlooked and can become a pathogen reservoir. To elucidate PWN infection status in asymptomatic trees, we performed PWN inspection of branches and trunks in 21 test trees in two different conditions: trees that had experienced PWN inoculation and those with suspected PWN infection that had experienced transient foliage discoloration. We detected PWNs in eight test trees (38%) and in 13 (1.5%) of a total of 843 samples. The difference in these percentages suggests that nematode inhabitation was highly localized within the trees, possibly owing to the restricted migration of PWNs. Our data demonstrated that trees that were once weakened but recovered their vigour can persist, as the asymptomatic carriers, in the forest. The implications for disease control are also discussed.     

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.     

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.     

A rapid staining‐assisted wood sampling method for PCR‐based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR‐based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining‐assisted wood sampling method aiding PCR‐based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5‐mg wood cross‐sections, of 0.5 × 0.5 × 0.015 cm dimension, that were cut from a wood sample of 0.5 × 0.5 × 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5‐mg wood cross‐sections (that adjoined the three 5‐mg wood cross‐sections, mentioned above) were cut. Nematode‐staining‐spots (NSSs) in each of the two stained sections were counted under a microscope at 100× magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus– specific amplicon of 403 bp ( DQ855275 ) was generated by PCR assay from 100.00% of 5‐mg wood cross‐sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx‐detection methods such as PCR assay.     

Direct PCR‐based method for detecting Bursaphelenchus xylophilus,the pine wood nematode in wood tissue of Pinus massoniana

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.     

A rapid,reliable and less‐destructive method for stem inoculations on trees

A new stem inoculation method using a gouge and syringe for inoculum application and utilizing the tree transpiration system for the uptake of sap‐compatible liquids was developed. This method is fast, reliable and less‐destructive compared to traditional methods of wound inoculation using a drill, punch, scalpel or a chisel.     

A simple method for measuring acetylene reduction of intact, nodulated black locust seedlings

A simple method is described for measuring acetylene (C(2)H(2)) reduction of nodulated black locust (Robinia pseudoacacia L.) seedlings. The C(2)H(2) reduction chamber consists of a standard plant pot sealed at one end with a stopper having holes for the seedling and a gas inlet port, and sealed at the other end with a stoppered collar with a gas port. The chamber temperature is regulated by circulating water at a controlled temperature through a copper coil surrounding the plant pot. A pump recirculates an approximately ten percent C(2)H(2) atmosphere through the system. Nine-week-old black locust seedlings, grown in sand culture and inoculated with Rhizobium, were used to obtain ethylene (C(2)H(4)) production curves. Ethylene production was linear for up to 60 minutes indicating that, for this particular symbiosis, (1) a simple closed system is adequate and (2) a short duration assay (10 minutes) will reliably estimate C(2)H(2) reduction over a longer period (up to at least 60 minutes). The water content of the soil medium had no influence on the C(2)H(2)/(C(2)H(2) + C(2)H(4)) ratio, which supports the suitability of C(2)H(2) as an internal standard for calculating C(2)H(2) reduction. This system has several desirable features. First, plant disturbance is minimized. Second, plants can be grown in inexpensive pots and no transplanting is necessary. Third, gas exchange and mixing are facilitated by a recirculating pump. Finally, root and nodule temperature can be controlled.     

A comparison of heat pulse velocity and lesion lengths for assessing the relative virulence of mountain pine beetle‐associated fungi on jack pine

The mountain pine beetle (MPB) vectors three blue‐stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to the success of the beetles and the death of the trees. The utility of two methods, heat pulse velocity (HPV) and lesion length, for assessing the relative virulence of these fungi were compared on jack pine in central Alberta. The HPV monitoring apparatus failed to detect xylem sap flow in any of the trees and, thus, could not be used to assess fungal virulence. In contrast, measurement of lesion lengths was more sensitive and provided further evidence that G. clavigera and L. longiclavatum are more virulent than O. montium. The failure of the HPV apparatus to detect sap flow suggests that the study trees were moisture stressed, a factor likely to increase their susceptibility to MPB. Thus, this method is not appropriate for assessing the response of the most susceptible (i.e. drought stressed) trees to MPB and its associated fungi.     

A loop‐mediated isothermal amplification‐based method for detecting Bursaphelenchus xylophilus from Monochamus alternatus

The introduction of the pine wood nematode (PWN), Bursaphelenchus xylophilus, to new areas has impacted on the international economy. Therefore, accurate and reliable detection methods for PWN are essential for the control and management of this pest. A rapid and economic method for detecting PWN may be developed focusing on the PWN vector (Monochamus alternatus). This work standardized a loop‐mediated isothermal amplification (LAMP) method using newly designed primer sequences based on the syg‐2 gene, which encodes the synaptogenesis protein syg‐2. Loop‐mediated isothermal amplification was conducted at 63°C for 60 min using six sets of primers. The result was confirmed by visual observation. A positive reaction was confirmed by SYBR Green I fluorescence dye under light thermal cycling. The lower limit of DNA detection was 51.4 pg/μl in both LAMP and 51.4 ng/μl in PCR. Therefore, the LAMP was 1,000 times more sensitive in DNA detection than PCR. The LAMP is a relatively new, highly accurate and rapid molecular technique that can rapidly detect infectious agents in the field without requiring sophisticated instruments, giving a visually readable result. The method greatly improves detection, without requiring professional knowledge and expensive, sophisticated equipment. Therefore, this system is suitable for quarantine and field detection.     

Reliability of field,greenhouse and cut‐shoot screening procedures for evaluating susceptibility of Scots pine to Melampsora pinitorqua

The reliability of field, greenhouse and cut‐shoot screening procedures for the assessment of the susceptibility of Scots pine (Pinus sylvestris) genotypes to Melampsora pinitorqua, the causal agent of twisting rust, was evaluated. Fourteen genotypes evaluated on half‐sib progenies after natural infection of 2‐year‐old plants showed comparable rank for rust susceptibility with those evaluated after controlled inoculation of 1‐year‐old seedlings in a greenhouse. The dynamic of pycnia production was assessed at 2‐day intervals in cut‐shoot assays under controlled conditions, giving reliable rankings of Scots pine genotypes between years and being significantly correlated with rust susceptibility assessed after natural infection of 2‐year‐old progenies. These results underlined the importance of some critical factors such as inoculum load and host phenology in the reliability of susceptibility evaluations assessed under different experimental conditions. Artificial inoculation of 1‐year‐old seedlings in greenhouse experiments could provide a useful early test for the management of Scots pine breeding programmes and study of inheritance of twisting rust susceptibility. However, in specific investigations the cut‐shoot assay would constitute a reliable laboratory test for studying host–pathogen interactions and the variability in pathogenicity of Melampsora pinitorqua populations.     

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 thirteen‐year study on the impact of a severe canker disease of Corymbia calophylla,a keystone tree in Mediterranean‐type forests

Worldwide, forests and woodlands have shown progressive declines in health as a result of global environmental changes in combination with local anthropogenic drivers. This study examined the incidence and progression of a canker disease of marri (Corymbia calophylla) caused by the endemic fungal pathogen Quambalaria coyrecup at three paired forest and anthropogenically disturbed sites in the southwest of Western Australia over 13 years. At the time of plot establishment in 2001, cankers were present on trees at all six sites with 22.7% of the assessed trees cankered. By 2014, cankers had led to the death of 6.7% of the trees, and an additional 10.0% of the trees developed cankers during this time. A further 2.3% of trees died due to causes other than canker, resulting in a final figure of 65.0% of trees remaining alive and free of cankers for the duration of the survey period. Canker incidence was significantly greater on trees present at anthropogenically disturbed sites (along roadsides and in paddocks) than forest trees (35.3% increasing to 50.7%, 10.0% increasing to 14.7%, respectively). Trunk diameter at breast height, tree height and crown ratings were not correlated with canker presence. This long‐term study provides evidence of the increasing severity of this canker pathogen and the impact it is having on the survival of marri.     

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.     

A multiplex PCR‐based technique for identification of Biscogniauxia mediterranea and Obolarina persica causing charcoal disease of oak trees in Zagros forests

Zagros oak (Quercus spp.) forests (ZOF) cover approximately 4 million hectares of the Zagros Mountains in Iran. Oak charcoal disease caused by Biscogniauxia mediterranea and Obolarina persica has recently increased in some regions of ZOF. Detection of these fungi in host tissue and identification of the anamorphs by traditional methods have limitations and difficulties which were overcome using two primers, OP1 and OP2, based on rDNA sequences of O. persica and used along with the specific primers MED1 and MED2 for B. mediterranea to develop a multiplex PCR. This method was used to correctly identify 1 pg of fungal DNA per 1 mg of inner bark tissues of Quercus brantii, Q. infectoria and Q. libani.     

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.