Perspectives on the use of plant growth promoting bacteria as biocontrol agents for the pine wilt disease

Pine Wilt Disease (PWD) has been considered one of the major threats affecting conifer forests and forestry economics throughout the whole world. This complex disease is caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus, through an intricate interaction with other partners like nematode associated bacteria and an insect vector that is responsible for nematode tree-to-tree transportation and dissemination. The first report of PWD dates to 1905 in Japan. Later, the PWD spread out to China (1982) and Korea (1988). In Europe, PWD was firstly reported in Portugal in 1999. Since then the disease has spread throughout the country and recently new PWD focus were found in Madeira Island (2008) and Spain (2010), thus indicating an elevated risk to other European countries (including the Scandinavia region) and forestlands. Although efforts for controlling and understanding PWD have been made, till now there are no significant solutions rather than the destruction of infected trees and forestlands, leading to huge losses, i.e 26 million m3 of timber in Japan, which is both economic and ecologically unacceptable. Therefore, new solutions are needed in order to control PWD. Some of the disease control methods developed rely on the use of chemicals with nematicidal effects. Plant breeding programs are also being conducted in order to obtain resistant pine trees. Yet, these approaches can endure as also they can be non-environmental friendly. In other plant diseases caused by plant parasitic nematodes the use of plant growth promoting bacteria (PGPB) as biocontrol agents is considered to be an interesting and effective green-approach. In this sense, the results obtained in our lab indicate that the inoculation of Pinus pinaster (maritime pine) seedlings with PGPB leads to a reduction of PWD development, as also to an increased plant growth. These results suggest that the inoculation of pine seedlings with PGPB in a “nursery” system can be a useful alternative in obtaining PWN resilient pine trees

Pine wilt disease and red ring disease of coconut: opportunities and perspectives in the study of two nematodes Bursaphelenchus xylophilus and B. cocophilus.

The xylophagous nematodes Bursaphelenchus cocophilus and B. xylophilus (Nematoda: Parasitaphelenchidae), are causative agents of two serious diseases, “red ring disease” (RRD) with great importance to coconut and certain oil palm species in Central and South America, and in “pine wilt disease” (PWD), which has wreaked havoc in forest areas in the Far East and now more recently in Europe (Portugal and Spain). PWD has not yet been detected in Brasil or any other country in Central or South America, but there is great risk of introduction due to worldwide movement of wood and wood products through major sea ports. Recently, for example, wood shipments originating from Brasil, infested with B. xylophilus, have been intercepted in China. Due to the taxonomical proximity between these two nematodes (the only two plant pathogenic species within the genus Bursaphelenchus, containing nearly 100 species), the similarity of life cycles (with the insect vectors Rhynchophorus palmarum for B. cocophilus and Monochamus spp. for B. xylophilus) and the likely genomic and proteomic similarities, a comparative study between these two nematodes may provide important insights into understanding RRD, in particular through the genome sequencing of B. cocophilus, and comparison with the recently sequenced genome of B. xylophilus. Knowledge on specific pathogenicity genes, involved in the RRD process, combined with past and present knowledge, should be helpful in designing new and effective control methods for this major tropical disease of coconut and palm

The pine wood nematode, Bursaphelenchus xylophilus, in Portugal: possible introductions and spread routes of a serious biological invasion revealed by molecular methods

The pine wood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a major world-wide pathogen and pest of pine, with impacts on forest health, natural ecosystem stability and international trade. In Portugal, PWN was first diagnosed in 1999, the first occurrence also for Europe. The disease was recently detected on the island ofMadeira and in northern Spain. In an attempt to search for more reliable and robust molecular markers that enable the study of intraspecific variability of B. xylophilus from different geographic locations, the intergenic spacer (IGS) region of the 5S rRNA gene and inter-simple sequence repeats (ISSR) analysis were used to determine the genetic relationships among 43 B. xylophilus isolates from Portugal, China, Japan, South Korea and USA. IGS sequence analysis showed that this region can only be used to establish interspecific relationships, since no differences were detected among Portuguese isolates from different geographic locations. Fingerprints obtained with ISSR show high genetic variability among Portuguese isolates, except for the ones obtained prior to 2008. The ISSR dendrogram suggests the spread of the disease inside continental Portugal and to Madeira. Until 2008, B. xylophilus populations found in continental Portugal showed low genetic diversity, pointing to a single introduction, probably from Asia, whereas recent populations from continental Portugal (2009-2010) and Madeira show high genetic diversity, suggesting multiple introductions from different origins

Are Bursaphelenchus xylophilus-associated bacteria playing a role in pine wilt disease?

Pine wilt disease (PWD), presently the most severe coniferous disease worldwide, is caused by the plant parasitic nematode Bursaphelenchus xylophilus, the pinewood nematode (PWN). Although PWN is considered the major pathogenic factor in PWD, its associated bacterial community is not ruled out as potential helpers in this complex and still little understood disease. This work presents the characterization of PWN-associated bacteria and plant pathogenicity trials in the pine host Pinus pinaster. The 16S rRNA gene sequencing of PWN-associated bacteria revealed the presence of bacteria from two phyla Proteobacteria (Burkholderiales, Pseudomonadales, Enterobacteriales and Xanthomonadales) and Firmicutes (Lactobacillales and Bacilalles). Phenotypic characterization revealed the presence of a heterogeneous bacterial community associated with PWN, exhibiting plant pathogenic traits common in wilting diseases. Our results suggest the intriguing possibility that some PWN-associated bacteria may play a significant role in the development of PWD

Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS-PCR-based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD

The use of the ACC deaminase producing bacterium Pseudomonas putida UW4 as a biocontrol agent for pine wilt disease

Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is responsible for the devastation of worldwide pine forestlands. Until today, the only effective solution to this serious threat resides on the destruction of infected trees, which is both economic and ecologically unacceptable. The use of ACC deaminase-producing plant growth promoting bacteria has been shown to be a useful strategy in order to reduce biotic and abiotic constraints that affect plant health and development. In this sense, we report the use of the ACC deaminase-producing bacterium Pseudomonas putida UW4 as a potential biological control agent for pine wilt disease. An inoculation assay was performed in 3-4 months Pinus pinaster (maritime pine) seedlings obtained from a nursery in Portugal. The bacteria P. putida UW4 wild-type and ACC deaminase mutant strains were inoculated in the roots of pine seedlings followed by stem inoculation of B. xylophilus. The inoculation of the P. putida UW4 wild-type strain lead to a significant reduction of B. xylophilus induced symptoms. Moreover, regardless the inoculation with B. xylophilus, seedlings inoculated with P. putida UW4 also demonstrated an increased root and shoot development. In addition, P. putida UW4 ACC deaminase knockout mutant was unable to promote pine seedling growth or to decrease B. xylophilus induced symptoms. The results obtained indicate that the inoculation of ACC deaminase-producing bacteria in pine seedlings growing in a nursery system might constitute a novel strategy to obtain B. xylophilus resistant pine trees. This is the first report on the use of ACC deaminase-producing bacteria as potential biological control agents for tree diseases

Development of Bursaphelenchus xylophilus-specific microsatellite markers to assess the genetic diversity of populations from European forests.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934), Nickle (Nematoda: Aphelenchoididae) is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in the world. Its introduction and spread in new forest ecosystems have considerable consequences both economically and environmentally. Therefore, it is of crucial importance to identify its invasion routes, to determine the origin of new outbreaks and to understand the invasion process of this species to prevent further dissemination of the disease in Europe. In order to address these questions using population genetic approaches, we have been developing a set of PWN-specific microsatellite markers, usable in routine conditions at the individual level, thanks to multiplex PCR coupled with a fast DNA extraction method. Microsatellites were isolated from a genomic library using a procedure combining DNA enrichment and high throughput pyrosequencing as recently described by Malausa et al. (2011). Primers were designed for 71 and 23 perfect and compound microsatellites, respectively, 26 of which were experimentally validated so far. Among them, 18 markers exhibited polymorphism after several rounds of amplification tests. Preliminary results on a set of 190 nematodes from 13 populations indicate a very low level of polymorphism in PWN populations from Portugal and Madeira Island, compared to populations from the native area in North America. The genotyping of a wide collection of samples from Europe, Asia and North America is currently underway in the laboratory. Assessing the genetic diversity of populations indeed constitutes the cornerstone to determine whether the European invasive PWN populations are the result of a single or several independent events of introduction

Heterogeneous microbial community associated with Bursaphelenchus xylophilus

Pine wilt disease (PWD) is a complex disease integrating three major factors: the causal agent, the pinewood nematode Bursaphelenchus xylophilus; the insect-vector Monochamus spp.; and the host pine tree, Pinus sp. Since the early 80’s, the notion that another pathogenic agent involved, namely bacteria, may play a role in PWD has been gaining traction, however the role of bacteria in PWD is still unknown. The present work suggests the intriguing possibility that some B. xylophilus-associated bacteria may play a significant role in the development of this disease. This is inferred as a consequence of: (i) the phenotypic characterization, of a collection of 35 isolates of B. xylophilus-associated bacteria, in different tests broadly used to test plant pathogenic and plant growth promoting bacteria, and (ii) greenhouse experiments that infer pathogenicity of these bacteria in in maritime pine, Pinus pinaster. The results illustrate the presence of a heterogeneous microbial community associated with B. xylophilus and the traits exhibited by at least some of these bacteria appear to be related to PWD symptoms. The inoculation of four specific B. xylophilus-associated bacteria in P. pinaster seedlings resulted in the development of some PWD symptoms suggesting that these bacteria likely play an active role with B. xylophilus in PWD

The role of Bursaphelenchus xylophilus associated bacteria in pine wilt disease

Pine wilt disease (PWD) is a complex disease integrating three major factors: the causal agent, the pinewood nematode Bursaphelenchus xylophilus; the insect-vector Monochamus spp.; and the host pine tree, Pinus sp. Since the early 80’s, the notion that another pathogenic agent involved, namely bacteria, may play a role in PWD has been gaining traction, however the role of bacteria in PWD is still unknown. The present work suggests the intriguing possibility that some B. xylophilus-associated bacteria may play a significant role in the development of this disease. This is inferred as a consequence of: (i) the phenotypic characterization, of a collection of 35 isolates of B. xylophilus-associated bacteria, in different tests broadly used to test plant pathogenic and plant growth promoting bacteria, and (ii) greenhouse experiments that infer pathogenicity of these bacteria in in maritime pine, Pinus pinaster. The results illustrate the presence of a heterogeneous microbial community associated with B. xylophilus and the traits exhibited by at least some of these bacteria appear to be related to PWD symptoms. The inoculation of four specific B. xylophilus-associated bacteria in P. pinaster seedlings resulted in the development of some PWD symptoms suggesting that these bacteria likely play an active role with B. xylophilus in PWD

Evidence for the involvement of ACC deaminase from Pseudomonas putida UW4 in the biocontrol of pine wilt disease caused by Bursaphelenchus xylophilus

Abstract Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is responsible for devastation of pine forests worldwide. Until now, there are no effective ways of dealing with this serious threat. The use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (encoded by the acdS gene)-producing plant growth-promoting bacteria has been shown to be a useful strategy to reduce the damage due to biotic and abiotic stresses. Pinus pinaster seedlings inoculated with the ACC deaminase-producing bacterium Pseudomonas putida strain UW4 showed an increased root and shoot development and reduction of B. xylophilus induced symptoms. In contrast, a P. putida UW4 acdS mutant was unable to promote pine seedling growth or to decrease B. xylophilus induced symptoms. This is the first report on the use of ACC deaminase-producing bacteria as a potential biological control agent for a tree disease, thus suggesting that the inoculation of pine seedlings grown in a tree nursery might constitute a novel strategy to obtain B. xylophilus resistant pine trees