Disease complex in coffee involving Meloidogyne arabicida and Fusarium oxysporum

Coffee corky-root disease, also called corchosis, was first detected in 1974 in a small area of Costa Rica where the root-knot nematode Meloidogyne arabicida is the dominant species. An epidemiological study revealed a constant association between Meloidogyne spp. and Fusarium sp. in cases of corky root. No corky root appears to have been reported in association with Meloidogyne exigua , which is the prevalent root-knot nematode on coffee in Costa Rica. Fusarium spp. are often cited as components of disease complexes in association with nematodes. Combined inoculations using M. arabicida or M. exigua with Fusarium oxysporum under controlled conditions showed that only the combination with M. arabicida produced corky-root symptoms on Coffea arabica cvs Caturra or Catuai. Fusarium oxysporum alone was nonpathogenic. Meloidogyne exigua or M. arabicida alone caused galls and reduction in shoot height, but no corky-root symptoms. When cultivars susceptible and resistant to M. arabicida were studied under field conditions for 5 years, all the susceptible cultivars exhibited corky-root symptoms on 40–80% of their root systems. Cultivars that were resistant to M. arabicida but not to M. exigua showed no corky root. These observations lead to the conclusion that corky-root disease has a complex etiology, and emphasize the dominant role of M. arabicida as a predisposing agent to subsequent invasion by F. oxysporum . Consequently, genetic resistance to M. arabicida appears to provide an effective strategy against the disease.     

香蕉品种对根结线虫病的抗性鉴定

在5个香蕉栽培品种对香蕉根结线虫抗病性测试中明确,威廉斯-F和北蕉表现中感,威廉斯-Y、粉蕉和 红蕉表现高度感病。     

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species

Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These quarantine proteinic markers have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (A67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the A67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.     

Control of root-knot nematodes (Meloidogyne spp.) in Hungary1

Under Hungarian climatic conditions, root-knot nematodes, and other phytoparasitic nematodes, generally cause damage in certain plant growing areas. These pests are present in glasshouses throughout the country. In the field the nematodes occur in sandy soil in the vegetable-growing areas of southern and central Hungary. The following species of root-knot nematodes have so far been reported to occur in Hungary: Meloidogyne incognita, M. arenaria, M. hapla, M. javanica, M. incognita acrita, M. thamesi and M. naasi. There are 10 specific nematicides registered for nematode control in the country. These chemicals are mainly applied in glasshouses where their use produces most economic return. Nematoderesistant cultivars are only available in the case of tomato. The nematodes cause the heaviest damage on glasshouse cucumber.     

Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives

Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (≈600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU?≈?1,700 and LSU?≈?3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values ≥0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (≥700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here.     

Metabolic profiling in the roots of coffee plants exposed to the coffee root-knot nematode, Meloidogyne exigua

To understand the effect of nematode Meloidogyne exigua infestation on coffee plants, resistant and susceptible coffee seedlings were inoculated with second-stage juveniles of M. exigua, and root metabolites were studied for four time intervals at 0, 24, 48 and 96 h. During this important period for parasite establishment, the concentrations of phenols, carbohydrates, amino acids and alkaloids in the roots were measured, and hydrogen nuclear magnetic resonance spectra of the root extracts were used to identify and quantify other metabolites. One of the most striking changes was the concentration of fumaric acid on resistant plants, which varied from 59 μg g(of root)^?1 to 138 μg g(of root)^?1 during the first 24 h of the nematode inoculation. The same level of variation was observed much later (96 h) in susceptible plants. Similarly, formic and quinic acid concentrations increased more rapidly in the resistant plants compared to the susceptibles. Sucrose concentrations increased to 370 % in the first 48 h in the resistant plants but showed no significant variation in the susceptible plant. Besides, the concentration of alkaloids was much higher at 24 and 48 h in the susceptibles compared to the resistant plants. These results suggest that the higher production of sucrose as well as formic, fumaric and quinic acids, and the lower production of alkaloids by the resistant cultivar in the first 48 h after the nematode inoculation are associated with the resistance of coffee plants to M. exigua.     

根结线虫食道腺细胞表达的基因及其应用潜力

根结线虫是国际公认的最为重要的植物病原线虫,研究其食道腺细胞表达的基因是了解其寄生性和致病性的基础。近年来国际上已从根结线虫食道腺细胞分离基因22个,其基因产物主要包括β 1,4内切葡聚糖酶、果胶酸裂解酶、分支酸变位酶、钙网蛋白、类毒液过敏蛋白等。本文对上述工作的研究进展作简要评述,并对今后的应用潜力进行了讨论。     

Screening of common bean (Phaseolus vulgaris) for resistance against temperate root-knot nematodes (Meloidogyne spp.)

BACKGROUND: An important part of the production area of common bean (Phaseolus vulgaris L.) in Belgium is located on the sandy soils of the provinces of Antwerp and Limburg where Meloidogyne chitwoodi (Golden), M. fallax (Karssen) and M. hapla (Chitwood) are present. The host plant status of ten bean cultivars for root‐knot nematodes was determined by evaluating penetration, development and egg mass formation after inoculation with second‐stage juveniles. RESULTS: The tested cultivars were poor to good hosts for M. chitwoodi, non‐hosts or bad hosts for M. fallax and excellent hosts for M. hapla. Significantly fewer M. fallax were found in the roots, and their development was delayed. Penetration of M. hapla took place over a longer period than that of M. chitwoodi and M. fallax. The number of mature females of M. chitwoodi in cv. Polder 6 weeks after inoculation was no different from that in other cultivars, although fewer egg masses were found on this cultivar in the screening test. There was no influence of M. chitwoodi on vegetative growth of cv. Polder. CONCLUSION: The differences found in host plant status of bean cultivars stress the importance of a correct diagnosis of the Meloidogyne species in agricultural fields. Cultivar Polder showed potential as a trap crop for M. chitwoodi. Copyright © 2011 Society of Chemical Industry     

线粒体COI基因作为根结线虫条形码的适用性分析

为评估线粒体COI基因作为根结线虫(Meloidogyne spp.)DNA条形码的适用性,对收集的25个根结线虫群体的线粒体COI基因进行扩增、测序,检测其扩增和测序成功率。将测序获得的COI基因序列与Q-bank中下载的根结线虫COI基因序列共同进行比对分析,利用MEGA 6.0计算种内、种间遗传距离进行"Barcoding Gap"检验以及构建NJ系统进化树作聚类分析,检验COI基因对根结线虫的物种识别能力。结果表明:线粒体COI基因扩增和测序成功率极高,扩增成功率达96%,测序成功率为100%。种间平均遗传距离为0.126,远远超过种内平均遗传距离0.002的10倍,种内遗传距离与种间遗传距离存在明显的"Barcoding Gap"间隔区。系统发育分析显示,COI基因可有效分辨出本研究15种根结线虫中的10种,物种识别率约为67%。线粒体COI基因具有极高的扩增和测序成功率、较高的物种识别能力,且扩增片段长度适宜,符合物种鉴定DNA条形码基因的标准,可作为根结线虫候选条形码基因。     

我国南方地区主要根结线虫DNA变异的RAPD分析

 本试验用5组随机引物对来自我国南方地区的30个根结线虫种群进行RAPD分析,并从中筛选出多态性较好的引物12个。共扩增出179条DNA多态带,各供试种群间存在着丰富的遗传多态性。扩增结果表现出种间差异大于种内差异的共同趋势,这表明上述12个引物能够较客观地反映种群间亲缘关系的远近。北方根结线虫与另外3种线虫(南方根结线虫、爪哇根结线虫、花生根结线虫)的亲缘关系最远;在3种主要根结线虫中,爪哇根结线虫与南方根结线虫的亲缘关系相对较近。基于种群间的相似系数分析和应用UPGMA法构建的聚类树状图,显示出不同的根结线虫在较低的相似性系数范围聚类,而绝大多数种内的不同种群均以较高的相似性系数聚在一起,这与形态分类基本一致,反映了形态学分类的分子遗传本质,同时也表明了应用RAPD技术进行根结线虫亲缘关系分析和种类鉴定具有合理性和可行性。本文还对RAPD方法对南方根结线虫小种鉴定的可能性进行了初步探讨。     

Expression of resistance to the root-knot nematodes,Meloidogyne hapla andM. fallax,in wildSolanum spp. under field conditions

In 1995 two fields in the Netherlands, naturally infested withMeloidogyne hapla (Wageningen) andM. fallax (Baexem), were used to evaluate resistant and susceptibleSolanum genotypes under natural conditions. In April, genotypes were planted in circular microplots. Soil samples were taken and analyzed for the occurrence of second-stage juveniles every six weeks. From August onwards, large differences between resistant and susceptible genotypes in numbers of juveniles were found in the soil. For all resistant wildSolanum genotypes the level of infection in soil at the end of the growing season in October was equal to or lower than at the beginning. Glasshouse experiments were performed with the same genotypes and nematode populations (i.e. originally derived from these fields) and the results were comparable with the observations from the field. It is concluded that resistance, as selected in glasshouse trials, corresponds well with resistant behaviour in the field and that it is worthwhile to transfer the resistance from theseSolanum sources to commercial potato cultivars for successful control of root-knot nematodes.     

Development of a new management strategy for the control of root-knot nematodes (Meloidogyne spp) in organic vegetable production

The nematophagous fungus, Pochonia chlamydosporia (Goddard) Zare & Gams, has been investigated as a potential biological control agent for use in integrated pest management strategies for Meloidogyne incognita (Kof & White) Chitwood in vegetable crops. The release of the fungus as a biological control agent requires a diagnostic method to monitor its spread in the environment and to gain knowledge of its ecology. Only molecular methods are sufficiently discriminating to enable the detection of specific isolates of fungi in soil. A method to extract DNA from soil was developed to increase the efficacy of PCR-based diagnostic tests that use specific primers. A selected isolate of P chlamydosporia var catenulata was applied at densities similar to those that occur naturally in nematode-suppressive soils. The fungus significantly reduced nematode infestations in soil following a tomato crop, in a strategy that combined the use of the fungus with crop rotation. The survival of the fungus in soil was also examined in controlled conditions in which it remained in soil in densities significantly greater than its original application rate for at least 5 months. Hence, it seems that populations of this fungus may be built up in soil and have significant effects on the regulation of root-knot nematode populations.     

放线菌防治根结线虫的作用机制及研究进展

根结线虫病是全球性植物土传病害,每年造成巨大经济损失。传统的线虫防治方法因成本高、毒性大、线虫易产生抗药性、危害生态环境等原因不符合农业生产绿色可持续发展的理念。放线菌因其能适应各种生境,代谢产物丰富,且能通过诱导植物产生免疫防御,调节植物根部微生物区系,分泌多种酶等方式来安全高效地防治根结线虫病而广受关注和研究。本文主要综述了放线菌防治根结线虫的作用机制及目前所取得的研究进展,以期为开发、应用放线菌资源进行线虫生物防治提供帮助。     

Assessment of PCR-based tools for the specific identification of some temperate Meloidogyne species including M. chitwoodi,M. fallax and M. minor

Several conventional PCR tests have been developed for the identification of the European quarantine root-knot nematodes Meloidogyne chitwoodi and M. fallax but data are lacking for the evaluation of their performance in terms of sensitivity, repeatability, reproducibility and specificity against a large range of populations. This study evaluated the performance criteria of three conventional PCR tests recommended by the consensus diagnostic protocol for Meloidogyne chitwoodi and Meloidogyne fallax published by the European and Mediterranean Plant Protection Organization (EPPO): a species-specific PCR (IGS target), a SCAR PCR, and a rDNA ITS PCR-RFLP. Evaluation was carried out with DNA extracts from juveniles, males and females according to EPPO recommendations for test validation. A minimum of 34 populations of target and non target nematode species were tested to check the specificity of these three PCR assays. The three PCR tests were ranked according to their specificity (with regard to cross reaction with other nematodes species or genus) and their sensitivity (detection of a single juvenile or mixed with other species). The species-specific PCR proved to be more sensitive but less specific than the SCAR PCR. The PCR-RFLP enables the identification of several Meloidogyne species but profile analysis can be difficult when several species are present in the mixture. Specific PCR products and RFLP profiles were also observed for M. arenaria and M. enterolobii, and described for M. minor and M. artiellia.     

Penetration and reproduction of root-knot nematodes on cucurbit species

Cucurbits are often cultivated in rotation with Solanaceae in double-cropping systems. Most cucurbits have been described as susceptible to root-knot nematodes (RKN) but little is known on their relative levels of susceptibility. Because RKN species differ in rates of root invasion and reproductive traits, isolates of M. arenaria, M. incognita and M. javanica were compared on five cucurbit hosts in experiments run in a climate growth chamber. They included zucchini squash cv Amalthee, cucumber cv Dasher II, melon cv Pistolero, pumpkin cv Totanera and watermelon cv Sugar Baby. All cucurbits were susceptible to the three RKN isolates although M. javanica showed higher invasion rates, faster development and higher egg production than M. arenaria on the selected cucurbits. Apparent differences among cucurbits were primarily due to root invasion rates and formation of egg masses. Both Cucumis species (cucumber and melon) were better hosts for nematode invasion and reproduction than zucchini squash, followed by watermelon. Large invasion rates followed by small reproduction traits were linked to M. incognita on zucchini squash. Reduced invasion rates and egg mass formation along with delayed early development were shown on watermelon.     

Effect of abamectin on root-knot nematodes and tomato yield

BACKGROUND: Tomato growers in Shandong Province, China, commonly face heavy root‐knot nematode infestations. Current methods of control include cadusafos and methyl bromide (MeBr), but alternative methods are required because of the high toxicity of these pesticides and the ecological risk of their use. Therefore, abamectin soil applications were evaluated for their potential to control soil nematodes in a series of laboratory tests, greenhouse pot experiments and field trials. RESULTS: Laboratory tests showed that abamectin exhibited rapid knockdown of Meloidogyne incognita, with LC₅₀ and LC₉₀ values that were superior to those of cadusafos and averaged 7.06 and 21.81 mg L^?1. In the greenhouse pot experiment, soil applications of abamectin provided significant M. incognita control similar to that provided by cadusafos while maintaining excellent plant height and vigour. In the field trials, abamectin exhibited excellent control effects to nematodes while giving a higher tomato yield. There was a 19.3–39.0% yield increase from the various treatments compared with the control, and the best results were obtained from the highest dose of abamectin. CONCLUSION: The results of this study demonstrated that abamectin has the potential to be used as an effective alternative to MeBr and cadusafos for nematode control in tomato production in Shandong Province. Copyright © 2012 Society of Chemical Industry     

Factors affecting the efficacy of non-fumigant nematicides for controlling root-knot nematodes

Second-stage juveniles (J2) and egg masses of root-knot nematodes as well as root debris heavily infected by the latter were exposed for different periods of time to six different doses of the nematicides cadusafos and fenamiphos. The efficacy of the nematicides increased significantly with increasing exposure time. Both nematicides were more effective against J2, although they could not provide acceptable control of J2 inside egg masses or heavily galled root debris. The effect of different application strategies on the efficacy and persistence of certain nematicides was also assessed in a field study. Cadusafos, fenamiphos, fosthiazate and oxamyl were applied in field micro-plots either as a single full dose at the time of crop establishment or as multiple reduced-rate applications at 14-day intervals throughout the cropping period, and their efficacy and persistence were determined using bioassays and analytical studies. Fosthiazate was the most efficient nematicide studied, and this was mainly attributed to its long soil persistence. Oxamyl also provided adequate nematode control for the first 48-56 days after its application, regardless of the application method used and its relatively rapid field dissipation. Fenamiphos and cadusafos failed to provide adequate nematode control, although cadusafos was the most persistent of the nematicides tested. The failure of fenamiphos to provide adequate nematode control was mainly attributed to its rapid degradation by soil micro-organisms, which were stimulated after its repeated low-rate application at 14-day intervals. In contrast cadusafos failure was attributed to the inability of the nematicide to reduce nematode populations even at relatively high concentrations in soil.     

Molecular diagnostic key for identification of single juveniles of seven common and economically important species of root-knot nematode (Meloidogyne spp.)

A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita , M. javanica , M. arenaria , M. mayaguensis , M. hapla , M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described.     

一种准确测定土壤根结线虫种群数量的方法

为探索一种准确测定土壤根结线虫种群数量的方法,采用NaOCl消解附着在土壤病残体上的根结线虫卵囊与线虫常规离心技术相结合的方法,测试了不同NaOCl浓度、离心时间对根结线虫回收效率的影响,筛选出最佳测定条件,并与浅盘法、离心法、Byrd法等线虫常规分离方法进行了比较。结果表明,在9个NaOCl处理浓度中,1.0%~2.5%NaOCl处理对土壤根结线虫的回收效率显著高于3.0%NaOCl处理,达到71.6%~99.4%。在1.0%、1.5%、2.0%和2.5%NaOCl处理30 s后,分别离心2~5、2~3、2~3 min和2 min对根结线虫的回收效率明显高于其它时间处理,在每50 m L土壤平均接种976粒线虫卵的人工病土中,上述处理分离线虫数量分别达到804.3~930.0、810.7~838.7、843.7~867.0和820.7。本研究的改良方法对自然病土中根结线虫的分离数量分别是浅盘法、离心法和Byrd法的29.3、13.4和2.1倍,显著提高了土壤中根结线虫的分离效率,可准确测定土壤根结线虫种群的数量。