Keywords: Pinewood Nematode (Bursaphelenchus Xylophilus), Pine Wilt disease, Biology, ... B. cocophilus is responsible for âRed Ring Diseaseâ of coconut.
PINEWOOD NEMATODE BURSAPHELENCHUS XYLOPHILUS – BIOLOGY AND DURABLE CONTROL
Submitted by
Md. Saiful Islam Registration No. - 851220383130
Course: Host-Parasite Interactions (NEM-30306) Supervisor:
Dr. Jaap Bakker November, 2014
Table of Content Abstract………………………………………………………….………..….………iii 1. Introduction……………………………………………………….……….…….…..01 History……………………………………………………………..…….…...01 Biology (Morphology, Taxonomy, Life cycle and symptom or disease development)……………………………………………………… ..…….….02 Research aim……………………………………………………..……….….05 2. Control…………………………………………………………………...…………..05 Quarantine, phytosanitary measures and hygiene…………………….……………05 Bio-control of beetles…………………………………………………….………….06 Bio-control of nematodes……………………………………………….….………..06 Resistant breeding for pine trees…………………………………..……..…………07 Chemical control of PWD……………………………………………….……….….08 Consequences of climate change on the spread of PWN……………………...….09 Possibility to interference ……………………………………………...…………..09 (Host, Nematode, Vector and Eco-climate environment) 3. Conclusion……………………………………………………………..….……..….10 Summary points……………….…………………………………….….…..……..10 Future Considerations………………………………………………....……….…11 Acknowledgement…………………………………………………..….….……..…12 Bibliography……………...…………………………………………..….……….…13 Appendices…………………………………………………………….…………….17
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ABSTRACT After devastating vast areas of pine forests in Asian countries, PWN Bursaphelenchus xylophilus vectored by Monochamus beetles causes the pine wilt disease spread into European forests and is causing world-wide concern. The disease involves a very complicated triangle interaction system between the pathogenic nematode, its vector beetle and host pine species. To prevent the complicated pine wilt disease as of becoming a global threat to pine forests, more precise information on durable control over PWD is a precondition for limitation of damage. The control strategies for PWD include intensified plant quarantine, integrated control of vectors, agricultural measures and replanting with resistant Pinus species. Among them replanting with resistant Pinus species or other coniferous species is the most effective over PWD. However, the resistance during screening systems depends on the aggressiveness of PWN populations used and their pathogenicity to plants. During this review study, a noticeable relationship between disease incidence and environmental factors has been revealed that high temperature and low moisture have an influence in favor of the PWD. Alternatively, low temperature and high humidity conditions are in disfavor to disease incidence and development. Several effective bio-control measures has higher disease control efficacy against PWD which may leads to the desired durable control. Therefore, further research could focus on the interruption of the disease triangle to provide the awaited effective and durable control over PWD.
Keywords: Pinewood Nematode (Bursaphelenchus Xylophilus), Pine Wilt disease, Biology, Durable Control
Abbreviations:
PWN- Pine Wood Nematode PWD- Pine Wilt Disease EPPO- European Plant Protection Organization QTL- Quantitative Trait Loci
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1 Introduction History: The pinewood nematodes (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD) that infects pine trees, is a serious pest and pathogen of forest tree species, unambiguously among the genus Pinus. This nematode causes PWD was first identified and reported from Japan in 1905 (Suzuki, 2002). Though, it has been devastating Japanese pine forests since the beginning of the twentieth century but until 1971 most researchers supposed that it might be due to the attack by pine beetles because the dead pine trees were almost always infested with abundant beetles larvae (Iwasaki and Morimoto, 1971). Actually, in 1968, a forest pathologist, Dr. Y. Tokushige, noticed that something moving on the surface of a petri-dish in which he was incubating pieces of wood from a dead pine tree. When he observed the petri-dish with a stereomicroscope, Dr. Tokushige saw that the moving organisms were a kind of nematode may be an alternate pathogen he suspected (Kiyohara and Tokushige, 1971). And yet, were doubtful that the nematode could be the causal organism and rapidly kill pine trees. At that time, they inoculated few pine trees with nematodes and within a few months all inoculated pine trees were wilted and lastly died. Consequently, Tokushige and Kiyohara carried out a large scale inoculation trial and confirmed about the pathogenicity of PWN and shown to be the causal agent of PWD (Kiyohara and Tokushige, 1971). Later they described the nematode as Bursaphelenchus lignicolus (Mamiya and Kiyohara, 1972). In the mean time it was spread into the East-Asian countries and reported from China, Korea and Japan in 1980s. In USA the PWN was first reported in Florida in 1934 associated with fungi in timber but first reported to induce PWD in 1979 (Steiner and Buhrer, 1934). Then, an extensive survey revealed the widespread distribution of the nematode throughout the USA ( Dropkin et al., 1981). In 1999, the disease was first detected and reported in Europe in Portugal (Mota et al., 1999) and is now a potential threat to pine forests worldwide. All the historic events related to PWD and researches are presented at a glance for quick overview below in (Appendix I).
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Biology: (Morphology, Taxonomy, Life cycle and symptoms and disease development) However, species of the genus Bursaphelenchus are difficult to distinguish as they are similar in morphology. Bursaphelenchus xylophilus is distinguished by three characteristics from other species of: i) in male the spicule is flattened into a disc-shaped cucullus at the tip, ii) in female the front vulval lip is distinct overlapping flap-like, and iii) the tail or posterior end of the body of the female is rounded (Smith et al., 2011). Though, B. xylophilus shows the general characters of Bursaphelenchus spp.: i) lips high and offset; ii) weakly developed stylet with reduced basal knobs and iii) median bulb well developed; dorsal oesophageal gland opening inside median bulb. But then again, especially in the female, the post-uterine sac is long and in the male, a) the tail is curved ventrally, b) conoid and has a pointed terminus, c) a small bursa is situated terminally and d) the spicules are well developed, with a prominent rostrum (Smith et al., 2011). The genus Bursaphelenchus has approximately 100 species (Vicente et al., 2011; Hunt, 2008 and kanzaki, 2008). Among all of them, only two are considered as plant parasitic, B. xylophilus and B. cocophilus. B. cocophilus is responsible for ―Red Ring Disease‖ of coconut and palm trees in Central and South America (Ryss et al., 2005). In 1937 Fuchs established the genus Bursaphelenchus includes nematodes which associated with insects and dead decaying trees predominantly conifers (Fuchs, 1937). The life cycle of PWN (B. xylophilus) comprises both phytophagous and mycophagous development phase (Vicente et al., 2011). Then again, most Bursaphelenchus spp. is mycophagous and is transmitted to trees during oviposition of insect vectors or by feeding by the insect vectors (Vieira, 2004) (Figure 1). However, other body parts of insect vectors such as elytra (wing case) and tracheae (breathing tube) could also transport the PWN. Mostly beetles i.e. Long-horned beetles from the genus Monochamus (Order: Coleoptera and Family Cerambycidae) are the vectors of PWN (Sousa et al., 2001, 2002).
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Figure 1. Life cycle of Bursaphelenchus xylophilus (Provided by P. Naves, EFN-INRB, Portugal)
The PWN life cycle has four propagative juvenile stages (J 1, J2, J3 and J4), latterly which moult to adult stage (Figure 1). The first juvenile stage (J1) is complete inside the egg and the second juvenile stage (J2) proceeds through hatching and then three (3) moults before turn into adults (Mamiya, 1984, 2004). Under favourable conditions, approximately at 20 0C the females of PWN lay 80-150 eggs during whole oviposition period of 28-30 days approximately and complete its life cycle from eggs to adults within 6 days (Appendix II). The interesting part is of the PWN life cycle is it varies with temperature as required 3 days at 30 0
C, 4 days at 25 0C, 6 days at 20 0C and 12 days at 15 0C. However, at temperature higher than
33 0C and at lower than 10 0C there is no reproduction take place (Mamiya, 1984, 2004). The PWN moults to adults through two different phases ―reproductive‖ or ―dispersal‖ life cycles and first two juvenile stages (J1 and J2) are common to both of them. In favourable conditions the nematodes moults to third and fourth juvenile stages (J3 and J4) and to finish reaches to the adult stage through reproductive pathway (Figure 1).
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The nematode require the dispersal pathway for surviving unfavorable conditions such as, i) low temperature, ii) lack of moisture and iii) lack of food. The third and fourth juvenile stages then referred to as JIII and JIV (Mamiya, 1984, 2004). This dispersal JIII and JIV juvenile stages are dissimilar in its morphological and biological features from reproductive J3 and J4 juvenile stages. This dispersal stage nematode larvae molt in wood and have specific features such as, i) a dome-shaped head, ii) sub-cylindrical tail, iii) degenerated esophagus and iv) lack of stylet. Most of the species from genus Bursaphelenchus are associated with insect vector cerambycid beetles. Monochamus spp. known as the pine sawyer beetles plays vital role for dispersal of PWN (Mamiya, 1984). Among the genus M. alternatus is the major vector for Asian countries (Mamiya, 1984; Akbulut and Stamps, 2011) and M. galloprovincialis for European countries especially in Portugal (Sousa et al., 2001, 2002). The insect vectors carried JIV larvae and hence easily transmitted to healthy new pine trees. For the transmission they (JIV stage nematode larvae) surround the pupal chamber and enter in body of the adult beetle through abdominal spiracles and are held in tracheae before the emergence of the pine sawyer beetle in wood (Mamiya, 1984). However, it is important to know that one adult beetle fly a maximum distance of 2.5 km approximately and carries around 0.3 million nematodes. On the other hand, under favourable condition J III larvae soon molt to J4 reproductive stage larvae and multiply rapidly (Mamiya, 1984). Generally, transmission of nematodes occurs through the feeding of young immature beetles after their emergence when they feed on healthy pine trees and enters in the host body through wounds done by feeding (Mamiya, 1984). Another important mode of transmission is oviposition of female insect beetles. On the other hand, when the female beetle lay eggs on dead or weakened pine trees then the JIV dispersal larvae renounce the insect body and enters the dead or weakening host through the site of oviposition wound (Akbulut and Stamps, 2011). In recent times, Zhao et al., 2000 and 2003 reported that PWD is a result of an integrative double vector system, where nematodes transmitted the pathogenic toxin producing bacteria within trees and the beetles the nematodes from tree-to-tree. This asssociation between PWN and phytotoxin producing bacteria is somehow similar to symbiotic association (Zhao et al., 2006 and 2007). Bacterial genus Pseudomonas and Burkholderia are considered abundant in association with B. Xylophilus (Vicente et al., 2011). However, further studies are needed to understand the specific role of this toxin producing bacteria’s on the complex PWD.
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In case of symptom and disease development PWD is characterized by a unique symptom of a reduction of trees oleoresin flow due to destruction of epithelial cells around resin canals by means of the invasion of nematodes (Mamiya, 1984). Simultaneously, it increases the ethylene production. Hence, at the onset of the advance stage prominent visible symptom appears by way of severe chlorosis via browning of the needles due to collapse of photosynthesis and water deficiency as a result of blocking the water movement by cavitation of tracheids in xylem of vascular system.
Research aim To know and study about considerable progress has been done in understanding the relationships between PWN, its vector, the host tree and environmental factors that results pine wilt disease worldwide.
2 Control Quarantine, Phytosanitary measures and hygiene: Despite the dedicated and concerted actions of government agencies, PWD continues to spread. World-wide trade of wood and wood products e.g. timber, wooden crates, palettes, etc., plays a vital role by means of potential dissemination ways of the PWN. B. xylophilus and its vectors are listed as red alert quarantine pests by EPPO (OEPP/EPPO, 1986). So far till date it is impossible to control PWN (B. xylophilus) if once entered into the tree. Thus, in case of cultural practice concentrated on combination of removal of PWD symptomatic and dead trees as soon as possible. In addition, control of vector beetles by insecticide treatment. Infected trees should be burned or buried after removal to prevent their use by means of a source of further infection and then PWD. It is important that the stumps also be removed, as that stumps are attractive to the sawyer beetles (Smith et al., 2011). Several phytosanitary treatments for wood logs and wood products have been proposed, for instance steam treatment, heat treatment, fumigation etc. in transit with phosphine. However, such treatments possibly will be expensive in relation to the price of the commodity. Hence, in 1986 EPPO recommends a standard phytosanitary measures for Europe and worldwide. Focal points of EPPO’s recommendations of phytosanitary measures is to prevent PWN and its vectors which covers plants and wood of all conifers from infested countries should be prohibited. If not possible to prohibit, wood products must have been heat treated to 5
a core temperature of 56 0C for 30 minutes (OEPP/EPPO, 1986). For the purpose of packing wood kiln drying could be acceptable and alternative fumigation should be required for particle wood. However, among all treatment methods heat treatment seems to be the only known most effective for wood products those are already infected with PWN and its vectors (Smith et al., 2011). Through the process of heat treatment all parts of wood products should reach a temperature of 56 0C for 30 minutes using commercial kiln practices (Smith et al., 2011 and OEPP/EPPO, 1986). Bio-control of beetles: From what is known so far, the nematode cannot infect a new tree without help of the vector. The early detection and felling of pine sawyer galloprovincialis infested pine trees would be an important step forward to prevent spread of PWN. Several efficient traps and attractants in combination with the cross-vane transparent trap with turpentine and ethanol lures are effectively used now days successfully used to control the vector (Monochamus beetle) of PWD (G. Álvarez et al., 2014). On the other hand, aerial spraying of insecticide is another effective way to control the progress of PWD by eradication of the insect vector. Management of the insect vector is the comprehensive line of attack to deal with PWD. Almost immediately after PWN detection in Portugal, successful attempts were made to lure beetles in combination with efficient traps which supposed to be a powerful tool for PWD management (Álvarez et al., 2014). Among those attempts two trapping system was notable i) The multiple-funnel trap, consisting of a series of black plastic funnels arranged vertically over a collection cup (Álvarez et al., 2014). ii) The cross-vane trap, consisting of cross-wed plastic vanes suspended vertically over a large funnel and collection cup (Álvarez et al., 2014). According to literature and practical experiences it is clear that efficient trapping system provide the key information for vector monitoring and also about by what means the nematodes being carried (Álvarez et al., 2014). This management of the insect vector may also work for direct control of the pine sawyer population.
Bio-control of nematodes: To control PWD through traps, insecticides etc. against the insect vector have very low efficiency. On the other hand, tree vaccination with nematicide, selective breeding of resistant trees etc. these methods are extremely labour intensive and expensive (Nunes da Silva et al., 2014). Development of sustainable, cost-effective and environmentally friendly methodologies is urgently needed due to the rapid spread of the disease and the severity of its consequences. 6
Chitosan is a deacetylated polysaccharide which is derived from chitin and obtained from the outer shell of crustaceans and cell walls of certain fungi (Nunes da Silva et al., 2014). It is shown in several studies that chitosan can induce plant resistance to numerous pathogens i.e. root-knot nematode, where the mode of action is restriction of pathogen growth by stimulating several defense mechanisms (Nunes da Silva et al., 2014). According to Nunes da Silva et al., 2014, in their study reduced PWN reproduction inside stem tissues was observed due to the impairment of reproduction ability and also the induction of physiological alterations in both nematodes and plants (Khalil and Badawy, 2012), which inhibits the successful reproduction of the nematodes.
Esteya vermicola is a bio-control fungus against PWD caused by PWN (B. xylophilus) (Xue et al., 2014). The lunate conidia produced by E. vermicola, have adhesive properties and could attach to the cuticle of nematodes hence, causing subsequent infections (Xue et al., 2014). Actually, lunate conidia germinate after the adherence and form a sharp infection peg to penetrate the cuticle of PWN. Then the fungus consumes the content of the infected nematode’s body and after consumption grows out from its dead body, and then again produce new lunate conidia’s for the next infection cycle (Xue et al., 2014). In a nutshell, the whole bio-control process is a subsequent infection cycle and it has great potential as a green biological control agent against PWD.
Resistance breeding for pine trees: Natural resistance is one of the most effective means to control majority of pests and diseases. For the reason, resistance that prevents plant-nematode interaction are most desirable such as, a resistant plant prevents multiplication of nematode population (Trudgill, 1991). In resistance breeding usually two main strategies are used: selection and cross breeding. Selection breeding programme is common in China, Japan and other North-Asian countries (Ohoba, 1976). Naturally occurring PWD resistant plants from forests where there are normally susceptible plant species grown are exploit in this selection programmes. Though, such resistance plants are rare. The major advantage of selection method is that it enables to start with a mature tree with desirable traits such as different structure of the resin canal which inhibit normal migration of nematodes and so, symptom or disease development (Kuroda, 2004). Contrary, the main challenge in breeding for natural resistance is the time requires for the maturity of trees to reach the desirable traits to be assessed for the resistance.
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Then again, cross breeding is more likely to identify genuine resistance found in tree species with natural resistance against PWN. Generally, in cross breeding desirable characteristics are introgressed into cultivars by repeated crossing and backcrossing (Nose and Shiraishi, 2003). However, there is an exception in this case of forest trees. Here, the F1 lines are directly put to use (Nose and Shiraishi, 2003). In case of marker assisted cross breeding the major advantage is it can shorten the breeding period but then the complexity of the resistance has been identified by selection procedure which is usually controlled by QTLs rather than a single dominant R-genes (Nose and Shiraishi, 2003). Moreover, tissue culture technique can be used to speed up the cross breeding programme and multiplication of selected natural resistant lines (Peng and Moens, 2003).
Chemical control of PWD: A number of Japanese and Chinese researchers reported that the phytotoxin producing bacteria which is associated with the PWN are involve in the development process of PWD (Kwon et al., 2010). Specifically, the bacteria promote pine wood nematode reproduction and the bacterial growth significantly increase by the presence of pine wood nematode (Kwon et al., 2010). However, actual mechanism of the disease in relation to producing bacteria has not been clearly explained. Several scientists reported that, the bacterial association with the PWN plays an important role in pathogenicity development. This detection makes sense that PWD is induced by means of combination of PWN and associated phytotoxin-producing bacteria hence, suggest that antibacterial agents could suppress PWD. According to Kwon et al., 2010, Oxolinic acid, known as an antibacterial agent strongly inhibits most of the bacteria isolated from pine wood nematodes. Though, Oxolinic acid is virtually inactive against B. xylophilus, but the interesting fact is that it effectively suppresses the development of PWD in greenhouse and field conditions. This result indicates that PWD can be controlled effectively by antibacterial agents though the definite mechanism is not clearly known. Besides Oxolinic acid, a combine mixture of abamectin and oxolinic acid has higher degree of disease control efficacy against PWD compare to single application of either abamectin or oxolinic acid (Kwon et al., 2010). On top, oxolinic acid, a synthetic chemical, is known to be very cheap compared with abamectin. On the other hand, solitary use of antibacterial agents is not recommended since aseptic pinewood nematodes may be remaining in healthy pine trees can cause PWD whenever they meet phytotoxin producing bacteria in future (Kwon et al., 2010).
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Consequences of climate change on the spread of PWN Empirically, this serious PWD generally associated with high temperature and so, occurs only where average summer temperature exceeds 20 0C. However, in combination with high temperature PWD is more frequent and destructive where there is little rainfall. Hence, changing climatic condition worldwide due to various causes for instance global warming due to deforestation, carbon increasing etc. would favor the spread of PWD. On the other hand, for the period of disease progress, water status of the host (pine trees) plays the vital role in the relationship between pine tree and pine wood nematode. According to Suzuki (2002) ―two factors, physiological water status and nematode population density are considered to be the decisive factors in development of PWD‖. Possible interference and control strategy relating to the physiological water status in pine trees may provide a valuable durable control over PWD as well as provide an insight over consequences of climate change since both temperature and water status of the host trees are correlated with climatic conditions. Possibility to interference for durable control: To control PWD management practices are concentrated merely limited on preventing the spread of nematodes. In these circumstances, possible interference on life cycle of PWN, vector and host will be alternative means of control to prevent PWD worldwide by breaking the disease triangle between pine tree, pine sawyer beetle and pine wood nematode. Several aspects of the insect’s biology (such as longevity, sexual maturation period fecundity and oviposition rates), life-history and interaction with the nematode and host should be studied to understand the actual stage or time of control which may have successfully prevent the spread of PWD. It has been found that pine sawyer beetle has only one generation per year (Dropkin et al., 1981) (Appendix III), with beetles emerging from the month of May to August in Portugal (Sousa et al., 2001, 2006). Possible interference and control measures strategy relating to the knowledge from vector insect’s biology may provide us the most challenging and valuable durable control over PWD. Hence, a better understanding over the inter-relationships between the nematode, its vectors and the host pine trees is clearly a precondition for limitation of damage by B. xylophilus.
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3 Conclusion PWD is the major threat to forest ecosystem and wood industry worldwide both environmentally and economically. However, traps, insecticides against the insect vector, tree vaccination with nematicide, selective breeding of resistant trees have very low efficiency and these methods are extremely labour intensive and expensive. Due to the rapid spread of the disease and the severity of its consequences, development of sustainable, cost-effective and environmentally friendly methodologies is urgently needed. In future a better understanding of all possible interference and control measures strategy relating to the knowledge from the complicated interactions and inter-relationships between the nematode, its vectors and the host pine trees is undoubtedly a precondition for decreasing the damage done by PWN (B. xylophilus). For the reason, knowledge’s about the interactions are presented in this review. Furthermore, highlights a number of potential breakthrough possibilities for the breakdown of the disease triangle to find out a durable control over PWD in summary points below-
Summary Points: The disease incidence is closely related to climatic conditions such as a high temperature with little rainfall as mentioned before. Life cycle of PWN involves two distinct forms: i) propagative forms under favorable conditions and ii) dispersal forms when conditions are not suitable which make the disease incidence very difficult to control. Quick wilt and death of pine trees by the nematodes due to the dysfunction of water conducting system through the death of parenchyma cells responsible water conductance. Several effective bio-control measures has higher disease control efficacy against PWD which may leads to the desired durable control however, they have very low efficiency, extremely labour intensive and expensive.
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Future considerations On the basis of above-mentioned information’s, it is clear that cool forested countries where summer temperature is not so high has natural immunity to PWD and its outbreak. Research over the past two decades has helped define the nematode-vector-host association and their roles in the pine wilt disease system. However, many questions remain unanswered and new questions continue to arise in response to recent advances in our understanding of this complex system. Therefore, further research could focus on the studies of chemical compounds which control the exodus of nematodes from beetles and the subsequent invasion into the tree. Hence, interrupts the disease triangle to provide the desired durable control over PWD a new strategy for disease control.
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Acknowledgement The author expresses his profound sense of thanks to Professor Dr. Jaap Bakker, for the interesting review topic and supervising the writing with a very cordial manner and critical evaluation of this manuscript, during the course Host-Parasite Interactions (NEM-30306), Laboratory of Nematology, Wageningen University and Research Center, The Netherlands.
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http://www.ncbi.nlm.nih.gov/pubmed/25002361 [Accessed February 18, 2015]. Zhao, B. G., Guo, D. S., and Gao, R. 2000. Observation of the site of pine wood nematodes where bacteria are carried with SEM and TEM. Journal of Nanjing Forest University, 24: 69–71. Zhao, B. G., Wang, H. L., Han, S. F., and Han, Z. M. 2003. Distribution and pathogenicity of bacteria species carried by in Bursaphelenchus xylophilus China. Nematology, 5: 899– 906. Zhao, B., Liu, Y., and Lin, F. 2006. Mutual influences in growth and reproduction between pinewood nematode Bursaphelen chusxylophilus and bacteria it carries. Frontiers of Forestry in China, 3: 324–327. Zhao, B., Liu, Y., and Lin, F. 2007. Effects of bacteria associated with pine wood nematode (Bursaphelenchus xylophilus) on development and egg production of the nematode. Journal of Phytopathology, 155: 26–30.
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Appendices Appendix-I. Chronological events in the history of PWN research
Source: (Guang et al., 2008)
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Appendix-II. Life cycle of Bursaphelenchus xylophilus (source: Oh et al., 2009)
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Appendix-III. Seasonal overview of the life cycle of B. alternatus
Season Spring Summer Fall and winter spring
M. alternatus Adult beetle carrying nematodes and flies to healthy pine trees. Beetle oviposits on dying pine trees. Larvae burrow under bark and moves towards deeper tissues. Insect pupates and molts to adult and so, mature feeding and flies to healthy pine trees.
*Adapted from (Dropkin et al., 1981)
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