Suppression of pine wilt disease by an antibacterial agent,oxolinic acid

BACKGROUND: Pine wilt disease (PWD) is very complex and has been reported to be caused by pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and its accompanying bacteria. However, there is no report on the control of PWD by antibacterial agent. The present study was performed to investigate disease control efficacy of antibacterial agents against PWD. RESULTS: Among six antibacterial antibiotics tested, oxolinic acid (OA) showed the strongest antibacterial activity against five bacteria isolated from three strains of pine wood nematode. In in vivo assay, it effectively suppressed the development of PWD in three‐year‐old seedlings of Pinus densiflora Sieb. & Zucc.; it showed 71% control when injected at 3 mg per seedling. A mixture of OA and the nematicidal agent abamectin (Ab) showed higher disease control efficacy against PWD than either OA or Ab alone. In addition, OA alone and a mixture of OA and Ab also controlled PWD in approximately 20‐year‐old pine trees under field conditions. CONCLUSION: This is the first report on the suppression of PWD by OA. The result strongly indicates that PWD could be controlled by antibacterial antibiotic alone and a combination of antibacterial and nematicidal agents. Copyright © 2010 Society of Chemical Industry     

Sublethal infection of different pine species by the pinewood nematode

Bursaphelenchus xylophilus, the pinewood nematode and causal agent of pine wilt disease (PWD), is a globally destructive pine pest. PWD is favoured by high temperatures and drought, but B. xylophilus can also persist in symptomless hosts in areas not prone to PWD. We compared seedlings of different Pinus species that were inoculated with B. xylophilus when well-watered and under moderate temperatures, conditions that favour sublethal infections. We compared isolates of B. xylophilus in south-west Europe (Portugal) and south-east USA (Louisiana). P. pinea, P. pinaster, and P. radiata were challenged in both areas with inoculations of local B. xylophilus. P. sylvestris in Portugal and P. palustris in Louisiana were also tested. Seedling mortality was low, as expected, but nematode establishment was common. There were higher nematode densities and more damage in seedlings of P. sylvestris and P. radiata, followed by P. pinaster, with P. pinea and P. palustris proving to be more tolerant. Life history of hosts was a better predictor of resistance to B. xylophilus than geographical origin, even under sublethal conditions. Fast-growing species (P. radiata and P. pinaster) appeared most likely to be symptomless carriers in areas not prone to the development of PWD. Cold-adapted species (P. sylvestris) may still be suitable hosts for the pathogen, even at the sublethal conditions of their distribution areas. There were more nematodes per gram of host using nematode isolates from Portugal. Nonetheless, our results opposed the hypothesis that North American pinewood nematodes have such low virulence that they are obligatorily saprophytic.     

Susceptibility evaluation of Picea abies and Cupressus lusitanica to the pine wood nematode (Bursaphelenchus xylophilus)

Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28 days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well‐known PWN host. Furthermore, the stem ultrastructure of P. abies, C. lusitanica and Pn. pinaster was analysed by scanning electron microscopy. The results suggest that P. abies and C. lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P. abies seems to be a compatible host that could act as a repository for PWN.     

西藏油菜种子内生菌分离及具有多种促生特性的拮抗菌筛选

为保障油料作物安全和绿色农业发展,从西藏自治区4个市收集5种甘蓝型油菜种子样品,通过平板分离获得种子可培养内生菌,对获得的菌株进行溶磷、解钾、固氮和产吲哚乙酸(indoleacetic acid,IAA)促生特性测定,筛选具有多种促生特性的菌株,测定其对禾谷镰孢菌Fusarium graminearum、油菜菌核病菌Sclerotinia sclerotiorum和燕麦镰刀菌F. avenaceum三种病原菌的拮抗性能及对油菜幼苗的促生效果,并对其进行分子生物学鉴定。结果显示,西藏油菜种子可培养内生菌丰富,优势菌为芽胞杆菌,共分离到110株内生菌,其中具有多种促生特性的菌株为12株,有4株菌株对禾谷镰孢菌、油菜菌核病菌和燕麦镰刀菌中2种以上菌有抑制作用,抑制率在49.50%～66.83%之间,分别为DJ-T-6、NM-8-10、DJ-L-4和BL-T-15菌株,经16S rDNA基因序列鉴定其分别为贝莱斯芽胞杆菌Bacillus velezensis、萎缩芽胞杆菌Ba. atrophaeus和缺陷短波单胞菌Brevundimonas diminuta,且均对油菜幼苗有显著的促生作用。表...     

Emamectin benzoate as a candidate for a trunk‐injection agent against the pine wood nematode,Bursaphelenchus xylophilus

In order to develop an effective trunk‐injection agent against pine wood nematode, Bursaphelenchus xylophilus, an in vitro assay was used to examine the antinematodal activity of 58 commercially available compounds with known modes of action. Among compounds tested, the GABA receptor agonists had better anti‐nematodal activity than compounds influencing glutamate, N‐methyl‐D ‐aspartate, β‐adrenergic, dopamine, muscarinic acetylcholine and nicotinic acetylcholine receptors, as well as those inhibiting acetylcholinesterase, monoamine oxidase, 5‐hydroxytryptamine uptake and Ca²⁺, K⁺, Na⁺ and Cl⁻ channels. Avermectins and milbemycins strongly inhibited propagation of the nematode. Emamectin benzoate proved to be the most active (IC₉₅ 0.050 µM ) being over 140 times more active than the active ingredient of conventional trunk‐injection agents. It is concluded that emamectin benzoate is a strong candidate for an anti‐nematodal trunk injection agent. © 2000 Society of Chemical Industry     

Enhancement of sphaeropsis canker of aleppo pine by the Israeli pine bast scale

An interaction between the fungal pathogenSphaeropsis sapinea (Fr.) Dyko & Sutton, causal organism of Sphaeropsis canker, and the Israeli pine bast scaleMatsucoccus josephi (Homoptera: Margarodidae), causal agent of pine decline, was studied, since both were prevalent on Aleppo pine (Pinus halepensis) stands.Sphaeropsis sapinea was isolated from larvae (stage II) and molts of the Israeli pine bast scale collected in four Aleppo pine stands. When Aleppo pine saplings were infested with pine bast scale and subsequently inoculated withS. sapinea most plants died, whereas mortality of seedlings inoculated withS. sapinea alone without previous scale infestation was negligible. Scale infestation of saplings without subsequent fungal inoculation caused the death of one third of the plants. Wounding of pine shoot apices by removal of needle fascicles enhanced infection byS. sapinea. The results support the assumption thatM. josephi can play an important role in transmitting the fungal inoculum to the host tree as well as enabling its penetration to the shoots through wounds in the bark. http://www.phytoparasitica.org posting Dec. 20, 2004.     

Aleppo pine provenances vary in susceptibility and secondary chemical response to Gremmeniella abietina infection

Aleppo pine is a thermophilic species that has become a popular tree species in plantings even beyond its natural habitat. In 1999 the pathogenic fungus Gremmeniella abietina was isolated for the first time in Aleppo pine plantations in Spain. The main objective of this study was to analyse the variation in susceptibility to G. abietina (European race, biotype A) infections among trees from five Aleppo pine provenances. Artificial inoculations were performed at two different times in January 2012. The susceptibility of the trees was evaluated by recording disease severity and the length of internal stem necrosis produced by the pathogen. In addition, the concentrations of two flavanone compounds were quantified as putative indicators of resistance. Provenances and G. abietina isolates both significantly affected levels of necrosis although the results were dependent on the time of inoculation. There was a relationship between provenance and resistance, with trees of higher elevation provenances being more susceptible to disease than trees of lower elevation provenances. The amount of naringenin flavanone from trees was significantly different between provenances, suggesting that it is a possible indicator value for resistance. Conversely, no differences were found in the concentration of eriodictyol in trees between provenances. The results show that the provenance‐dependent variation in the susceptibility of Aleppo pine to G. abietina should be considered in selection of seed sources for restoration.     

基于灰色关联度分析气候因素对江西省松材线虫病的影响

为分析不同气候因素对松材线虫Bursaphelenchus xylophilus引起的松材线虫病的影响程度,本文以江西省全南县(代表赣南,松墨天牛一年2代)和德兴市(代表赣北,松墨天牛一年1代)遭受松材线虫病危害的马尾松林生态系统为研究对象,获取2018年-2021年松材线虫病普查数据和1990年-2021年的主要气象因子数据,结合滑动平均法和灰色关联度分析法比较分析两地的气候差异和疫情年间气候变化对松材线虫病发病面积的影响。结果显示:除德兴市的年均相对湿度和年降水量与历年平均值相比有所降低外,两地其他气象因子均呈上升趋势;灰色关联度分析表明,年均最高气温和年均风速是影响赣南松材线虫疫情的重要气象因子,年均最高气温和年降水量是影响赣北松材线虫疫情的重要气象因子;气温升高促进松材线虫和其媒介昆虫的生长繁殖与活动,风速升高可使媒介昆虫远距离传播松材线虫,降水量减少可降低松材线虫和媒介昆虫染病死亡几率。赣南赣北气候的整体变化趋势都有利于松材线虫病的扩散蔓延。     

Influence of rice development on the function of bacterial blight resistance genes

Disease resistance genes most commonly used in breeding programs are single, dominant genes with relative effectiveness that is sometimes influenced by plant developmental stage. Knowing the developmental stages at which a resistance gene is functional is important for disease management. In rice, resistance at the seedling stage is crucial, because wounding during transplanting increases the potential for bacterial blight disease, and not all bacterial blight resistance genes are effective at the seedling stage. Effectiveness of the bacterial blight resistance genes Xa4, xa5, and Xa7, all in a common genetic background, was evaluated at different developmental stages by measuring lesion length and bacterial numbers after inoculation with the bacterial pathogen, Xanthomonas oryzae pv. oryzae. The Xa4 and xa5 genes controlled disease at all growth stages. In contrast, Xa7 was not fully functional in very young seedlings, but was completely effective by 21 days after sowing (das). The effects of plant developmental stage on interactions of the Xa7 gene with X. oryzae pv. oryzae strains carrying different mutant avrXa7 alleles were also tested. If a partial or fully functional avrXa7 allele was present, Xa7 resistance was effective at all growth stages tested after the transplant stage (>21 das).     

注干用10%甲氨基阿维菌素苯甲酸盐可溶粒剂的研制及对松材线虫病的防治效果

为有效防治松材线虫病，研制了注干用10%甲氨基阿维菌素苯甲酸盐 (以下简称甲维盐) 可溶粒剂，并通过在马尾松树干基部钻孔施药，考察了其对松材线虫病的防治效果。以苯甲酸钠、单水乳糖、可溶性淀粉和羧甲基纤维素为水溶性载体，采用混料均匀试验设计及偏最小二乘回归分析法对水溶性载体的配方进行了优化。将水溶性载体与溶于正丁醇和乳化剂OP-10的甲维盐 (油相) 在不同时间下混合并造粒，通过对其性能指标的追踪检测得出粒剂最佳配比和混合时间。结果表明:10%甲维盐可溶粒剂的最佳配比 (质量分数) 为:甲维盐10.0%(折纯)，乳化剂OP-10 6.7%，正丁醇12.0%，苯甲酸钠39.0%，单水乳糖20.5%，水溶性淀粉3.0%和羧甲基纤维素4.5%；机械混合时间为10 min，粒剂外观为乳白色圆柱体，各项性能指标合格。注干施药结果表明:在浙江临安东湖村松材线虫病疫区，注干110棵马尾松，1 a后马尾松死亡率为0，对照区为6.1%；在福建泉州仙公山疫区，注干230棵马尾松，1 a后马尾松死亡率为1.7%，对照区10.8%。结果表明，所研制的10%甲维盐可溶粒剂可在松脂分泌旺盛的松树上注干施药，制剂造粒工艺简单，无包装物污染，对松材线虫病防治效果显著。     

Identification of differentially expressed genes in tolerant and susceptible potato cultivars in response to Spongospora subterranea f. sp. subterranea tuber infection

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) has recently become one of the most devastating potato diseases of economic importance in South Africa. The use of resistant cultivars has long been considered the most effective and sustainable strategy to manage the pathogen. However, little is known about the molecular mechanisms underlying resistance of potato tubers to Sss. Using RNA-sequencing (RNA-seq), 2058 differentially expressed genes (DEGs) were identified from two potato cultivars (tolerant and susceptible) in response to Sss infection. Analysis of the expression patterns of 10 selected defence-response genes was carried out at two different stages of tuber growth using RT-qPCR to validate the RNA-seq data. Several defence-related genes showed contrasting expression patterns between the tolerant and susceptible cultivars, including marker genes involved in the salicylic acid hormonal response pathway (StMRNA, StUDP and StWRKY6). Induction of six defence-related genes (StWRKY6, StTOSB, StSN2, StLOX, StUDP and StSN1) persisted until harvest of the tubers, while three other genes (StNBS, StMRNA and StPRF) were highly up-regulated during the initial stages of disease development. The results of this preliminary study suggest that the tolerant potato cultivar employs quantitative resistance and salicylic acid pathway hormonal responses against tuber infection by Sss. The identified genes have the potential to be used in the development of molecular markers for selection of powdery scab resistant potato lines in marker-assisted breeding programmes.     

Detection of Diplodia sapinea in Corsican pine seeds

Diplodia sapinea is one of the major pathogens of pines worldwide. Despite the putative critical importance of seed infection in the epidemiology of the disease, this aspect of the biology of the fungus is poorly known. Here, biological and molecular methods were developed for the detection of the fungus and applied to assess D. sapinea infection in Corsican pine seeds. A buffered medium containing tannic acid and malt extract as a nutrient base was the most efficient and selective for D. sapinea recovery. A molecular method based on DNA extraction with a commercial kit and specific amplification, including an internal amplification control, was developed. A high percentage of infection (57% positive isolations) was observed in seeds obtained from fallen cones in a Corsican pine stand with no apparent symptoms of D. sapinea. Seeds collected from trees in a seed orchard showing severe symptoms of dieback caused by D. sapinea had comparatively lower infection (38%). Moreover, very low infection levels (1–5%) were observed after the standard treatment used for seed extraction, which included heating at 40°C. Diplodia sapinea was not recovered from seedlings grown from infected seed lots submitted to water stress. Overall, results suggest that the risk of disease transmission by commercial seeds is probably low, but could be further reduced by thermotherapy.     

Colonization of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots by <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> promotes growth and enhances systemic disease resistance through jasmonic acid/ethylene and salicylic acid pathways

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.     

Identification and characterization of bacteria isolated from crown galls on stone fruits in Poland

Eighty stone fruit nurseries located in different regions of Poland were examined for the presence of crown gall affected plants. The disease was observed in 39 nurseries, and galls were sampled for bacterial isolation. Out of 1213 isolates, 409 were pre‐identified as Agrobacterium/Rhizobium spp. with 23S rDNA‐based multiplex PCR, and out of these, 315 were pathogenic when tested on sunflowers. Sequence analysis of three housekeeping genes (fusA, recA, rpoD) revealed that 366 strains belonged to Rhizobium rhizogenes, 23 to Agrobacterium tumefaciens species complex, and the rest of the strains were allocated to new phylogenetic lineages. Of these, the most numerous was the lineage allocated in the Pararhizobium genus. Positive results obtained from pathogenicity tests were generally in agreement with results obtained by PCR with primers complementary to T‐DNA except for two strains, which were positive for PCR but negative for the pathogenicity test. All detected Ti plasmids were nopaline‐type. Independent of their pathogenicity, 59% of tested strains were not sensitive to agrocin 84 in in vitro tests. Analysis of biochemical and physiological features distinguished 50 groups with different phenotypic profiles, but the tested traits were not consistent for strains classified to one taxon. This finding shows limited value of biochemical tests in identification procedures. The bacteria causing tumours were heterogeneous and strains classified to different taxa were found even in a single tumour.     

Growth environment and seedling age affect constitutive and inducible defence in radiata pine

There is increasing interest in induced resistance for crop protection. This study investigated effects of growth environment on inducible defences in developing radiata pine (Pinus radiata) seedlings. Seedlings were grown in the presence or absence of Trichoderma in either a temperature-controlled growth room or in a greenhouse. After 3, 5 or 7 months, seedlings were sprayed with 2.25 mm methyl jasmonate (MJ), 1 week before quantification of monoterpenes and total phenolics. The magnitude of induction of monoterpenes and total phenolics by MJ was inversely proportional to constitutive content, was greater in the greenhouse than the growth room and was greater at 3 months than at 7 months. This may indicate a cost-saving strategy to limit defence induction once constitutive defence reaches a certain threshold. Growth room seedlings expressed greater constitutive resistance to terminal crook (Colletotrichum acutatum) and to diplodia dieback (Diplodia sapinea) than greenhouse plants, consistent with the relative differences in constitutive defence chemistry. Indeed, while MJ induced resistance to diplodia dieback in both environments, there was no difference in terminal crook incidence (4.1%) between MJ-treated and untreated seedlings in the growth room. By the end of the study, growth room seedlings had c. 70% more β-pinene in stems and 140% more total phenolics in needles than greenhouse plants but were 40% smaller, indicating a defence–growth trade-off. Trichoderma did not have a significant effect on defence chemistry or on pathogen resistance. These results demonstrate the potential to manipulate defence in radiata pine by modification of the growth environment.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

Native entomopathogenic nematodes isolated from Turkey and their effectiveness on pine processionary moth,Thaumetopoea wilkinsoni Tams

Two native entomopathogenic nematodes were isolated from soil samples in Eastern Mediterranean region of Turkey and characterized based on 28S rDNA region. BLASTN homology and phylogenetic analysis of SK17 and SK-71 isolates indicated 98% and 99% identity to Steinernema affine and Steinernema feltiae, respectively. The results were constructed by neighbour-joining and bootstrap tree methods. Efficacy of S. affine (SK-17 strain) and S. feltiae (SK-71 strain) was tested against the larvae of pine processionary moth, Thaumetopoea wilkinsoni Tams, and remarkable mortality rates were obtained. Both strains caused complete mortality upon application of 500 IJs in foliar tests. However, the same strains caused 30% and 33% mortality at 80 IJs/cm² in soil applications. It was concluded that these native strains could be considered as potential biocontrol agents for reducing the damage caused by T. wilkinsoni larvae.     

土传黄瓜立枯病高效拮抗菌的筛选鉴定及其生物效应

采用平板对峙法从黄瓜根际土壤中分离出的400余株细菌菌株中筛选出16株对立枯丝核菌Rhizoctonia solani具有拮抗效果的菌株,抑菌带直径在0.81～1.93 cm之间。并从中选出3株抑菌带直径在1.6cm以上的菌株N33、N35和N43,结合形态、生理生化特性及16S rDNA序列比对分析,鉴定N33菌株为假单胞菌属Pseudomonas sp.,N35和N43菌株为芽胞杆菌属Bacillus sp.。通过在黄瓜育苗基质中添加选育的高效拮抗菌株,观测其对黄瓜苗生长的促进作用以及对立枯病的防治作用,3株菌株均具有促进黄瓜苗期生长和防治苗期立枯病的作用,其中N43菌株促生及防病效果均最显著,地上部鲜重比对照处理增加62.16%,防治效果达62%。