Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Host–parasite relationships of Meloidogyne incognita on spinach

Host–parasite relationships in root-knot disease of spinach caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Nematode-induced mature galls were large and usually contained one or more females and egg masses with eggs. Feeding sites were characterized by the development of giant cells containing granular cytoplasm and many hypertrophied nuclei. The cytoplasm in these giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P _i) in a series from 0–128 eggs and second-stage juveniles per cm³ soil and growth of spinach cv. Symphony F₁ seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^P–T ] was fitted to fresh top- and total plant-weight data for inoculated and control plants. Tolerance limits ( T ) of spinach cv. Symphony F₁ to M. incognita race 1 for fresh top and total plant weights were 0·25 and 0·5 eggs and second-stage juveniles per cm³ soil, respectively. The minimum relative values for fresh top and total plant weights were zero in both cases at P _i ≥ 32 eggs and second-stage juveniles per cm³ soil. Root galling was least at low initial population densities and greatest at 16 eggs and second-stage juveniles per cm³ soil. Maximum nematode reproduction rate was 33·1-fold at the lowest P _i.     

Histological mechanisms of the resistance conferred by the Ma gene against Meloidogyne incognita in Prunus spp

The Ma gene from Myrobalan plum is a TNL gene that confers a high-level resistance to all root-knot nematodes of major economic importance, including Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The nematode behavior in the roots and the corresponding histological mechanisms of the Ma resistance to M. incognita in the resistant (R) accessions of the plum 'P.2175' and the interspecific hybrid P.2175×almond-peach '35', carrying the Ma1 allele (Ma1/ma), were characterized in comparison with the susceptible (S) accessions in the plum 'P.2032' and the interspecific hybrid P.2175×almond-peach '253' (ma/ma). Second-stage juveniles (J2s) were inoculated in micropropagated plantlets grown in soil substrate under controlled conditions at 25°C. Nematodes penetrated both R and S plants preferentially along the apical zone or close to the young lateral buds and moved via similar routes. Then they migrated into the cortex downward in the direction of the apex and turned up in the meristematic apical region to colonize the differentiating stele. In R accessions, motile J2s neither swelled nor developed into J3s, and initiation of feeding sites was never observed. This complete absence of gall symptoms is associated with cell necroses and corresponding hypersensitive-like reaction (HLR) phenotypes occurring either in the stele or in the meristematic apical region or in the cortex. Nematode attacks often disorganized the meristematic apical tissues of R accessions, which induced the development of subterminal lateral roots replacing primary terminal apices and, thus, provided an active resistance reaction to HLR damage.     

Penetration, Development and Emigration of Juveniles of the Nematode Meloidogyne arenaria in Myrobalan Plum (Prunus cerasifera) Clones Bearing the Ma Resistance Genes

Penetration, development and emigration of M. arenaria in the roots of three Myrobalan plum (Prunus cerasifera) clones genetically characterized for their resistance to root-knot nematodes (RKN) were studied during the 10 (penetration) and 15 (emigration) days following the date of inoculation (D) of 2500 juveniles (J2s) per plant into the soil. Miniaturized tests were conducted on the two resistant clones P.2175 (Ma1 gene) and P.1079 (Ma2 gene) and the susceptible clone P.2032 (recessive for both genes), obtained from micropropagated plantlets and grown in mini-containers under controlled conditions at 25°C in a growth chamber. For penetration and development studies, nematodes in the roots were recovered by the acid fuchsin-lactophenol staining technique. Equivalent numbers of J2s were recovered in all the clones at D+1 and D+2. Subsequently, the numbers increased rapidly in P.2032 and were significantly different from those in P.1079 and P.2175 that remained at a low level. No swollen larvae were observed in the resistant clones. In P.2032, the first swollen larvae were observed at D+4, the first females were observed at D+12, whereas the first females with attached egg sacs and the first new-generation J2s were obtained between D+21 and D+28. Our data suggest that the resistance phenomenon does not act on the very early nematode penetration but acts later by preventing feeding-site induction and development into the third-stage. For emigration studies, plants in which J2s had been allowed to penetrate for two days (from D to D+2) were washed free of soil, repotted and then, after various periods of growth, soil-free roots were placed under a mistifier to evaluate the numbers of emigrating individuals. Emigration of J2s from the roots occured mainly from D+2 to D+4 in all the genotypes and was very limited from D+4 to D+10. There was no significant differences in the number of emigrated juveniles between the resistant and susceptible clones, indicating that emigration cannot explain the difference in the numbers of nematodes recovered in the roots.     

Growth and photosynthesis of four invasive aquatic plant species in Europe

Crassula helmsii , Hydrocotyle ranunculoides , Ludwigia grandiflora and Myriophyllum aquaticum are four well known invasive aquatic plants in European waters. In this study, plant growth at different nutrient availabilities, regeneration capacity and photosynthesis were investigated. Results show high relative growth rates (RGR) of the species of up to 0.132 ± 0.008 g g⁻¹ dry weight (dw) day⁻¹ ( H. ranunculoides ) and a significant increase in RGR with increasing nutrient availability. All species show a high regeneration capacity and the ability to form new shoots from single nodes, even though it differs between the species. Ludwigia grandiflora and M. aquaticum also show regeneration from single leaves. Species differed in maximal amounts, and in temperature and light optima of net assimilation rates: H. ranunculoides leaves reach maximum photosynthetic rates of up to 3500 μmol CO₂ × h⁻¹ g⁻¹ dw, L. grandiflora (leaves) up to 2200 μmol CO₂ × h⁻¹ g⁻¹ dw, M. aquaticum (shoots) 400   μmol CO₂ × h⁻¹ g⁻¹ dw and C. helmsii (shoots) up to 200 μmol CO₂ × h⁻¹ g⁻¹ dw. Hydrocotyle ranunculoides , L. grandiflora and M. aquaticum preferred high light intensity and high temperatures, whilst C. helmsii was negatively affected by intense sunlight. Summarising, it can be assumed that at least H. ranunculoides , L. grandiflora and M. aquaticum can grow well under current and likely future central European climate conditions.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Variation in resistance to the root-knot nematode Meloidogyne incognita in tomato genotypes bearing the Mi gene

Root-knot nematodes ( Meloidogyne spp.) are among the main pathogens of tomato ( Lycopersicon esculentum ) worldwide. Plant resistance is currently the method of choice for controlling these pests and all the commercially available resistant cultivars carry the dominant Mi gene, which confers resistance to the three main species Meloidogyne arenaria , M. incognita and M. javanica . However the emergence of virulent biotypes able to overcome the tomato resistance gene may constitute a severe limitation to such a control strategy. To date, little was known of the possible influence of the homozygous vs heterozygous allelic state of the Mi locus, or the tomato genetic background, on the expression of the resistance. In order to test both these factors, the resistance was evaluated of a large panel of L. esculentum genotypes (selected from the Vilmorin germplasm stock collection) to seven M. incognita lines avirulent or virulent against the Mi gene. Plant resistance was estimated by counting the egg masses on the root systems after inoculation with second-stage juveniles (J₂). Reproduction of the nematodes was similar or, more often, significantly higher on heterozygous tomato genotypes than on homozygous ones, suggesting a possible dosage effect of the Mi gene. Data also indicated that the tomato genetic background had a major effect on the variations observed in nematode reproduction, especially when tomato genotypes were heterozygous for the Mi gene. These results have important consequences in terms of breeding strategies and durability of the resistance conferred by the Mi gene.     

Population differentiation in the banana leaf spot pathogen Mycosphaerella musicola, examined at a global scale

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of the global population of Mycosphaerella musicola , the cause of Sigatoka (yellow Sigatoka) disease of banana. The isolates of M. musicola examined were grouped into four geographic populations representing Africa, Latin America and the Caribbean, Australia and Indonesia. Moderate levels of genetic diversity were observed for most of the populations ( H  = 0·22–0·44). The greatest genetic diversity was found in the Indonesian population ( H =  0·44). Genotypic diversity was close to 50% in all populations. Population differentiation tests showed that the geographic populations of Africa, Latin America and the Caribbean, Australia and Indonesia were genetically different populations. Using F _ST tests, very high levels of genetic differentiation were detected between all the population pairs ( F _ST > 0·40), with the exception of the Africa and Latin America-Caribbean population pair. These two populations differed by only 3% ( F _ST = 0·03), and were significantly different ( P  < 0·05) from all other population pairs. The high level of genetic diversity detected in Indonesia in comparison to the other populations provides some support for the theory that M. musicola originated in South-east Asia and that M. musicola populations in other regions were founded by isolates from the South-east Asian region. The results also suggest the migration of M. musicola between Africa and the Latin America-Caribbean region.     

烟草品种资源对根结线虫病抗病性鉴定

 在1996和1997年,在温室采用卵接法和病土接种法,测定65份烟草种质资源对南方根结线虫1号小种和爪哇根结线虫的抗病性。其中,16份抗南方根结线虫1号小种,25份中抗南方根结线虫1号小种;仅有Va102中抗爪哇根结线虫;经田间试验测定10份烟草种质资源对花生根结线虫2号小种的抗病性,CF954对花生根结线虫2号小种表现中抗。本文还研究了卵接法和病土接种法对测定品种抗性的差别,结果表明二者无显著差异。     

Interactions between Fusarium oxysporum f. sp. racheiphilum and Meloidogyne spp. in Vigna unguculata. 2. Specificity of different taxa

A California isolate of Meloidogyne javanica increased Fusarium wilt symptoms in cowpea cultivars California Blackeye No. 3 (CB3) (resistant to wilt) and Grant (tolerant) inoculated with each of the three races of Fusarium oxysporum f. sp. tracheiphilum. The same isolate of M. javancia did not similarly increase wilt in wilt-resistant cultivar CB7977 inoculated with two isolates of race 3 of F. o. tracheiphilum. Six of seven isolates of M. javanica caused similar increases in vascular discoloration in cultivar CB3. but one isolate of M. javanica and seven of M. incognita did not. Vascular discoloration rating was positively correlated with galling severity. However, increasing the initial inoculum density, and thus galling index, of one isolate of M. incognita did not increase vascular discoloration. The vascular discoloration ratings for the wilt-susceptible CB5 controls in each experiment were higher than those for the wilt-resistant cultivars infected with M. javanica. It is hypothesized that M. javanica but not M. incognita reduces, but does not eliminate, resistance to all races of F. o. tracheiphilum in cultivars CB3 and Grant.     

南方、爪哇和花生根结线虫的快速灵敏的PCR鉴定方法

 为了研制南方、爪哇和花生根结线虫快速灵敏的检测和鉴定方法,分别分离了4个南方根结线虫和3个爪哇根结线虫特异性的随机扩增多态性DNA (RAPD)片段。在这些RAPD标记DNA序列的基础上,设计了多对SCAR PCR引物,并用源于国内外的南方、爪哇、花生、北方和象耳豆根结线虫群体验证其扩增特异性和灵敏度。最终确定了3对高效扩增的SCAR引物,它们组合使用可以可靠灵敏地鉴定南方、爪哇和花生根结线虫。3对引物的扩增灵敏度达1/3条的二龄幼虫、雄虫或雌虫,这表明本研究研制的PCR鉴定法可用于生产实践中土样和根样中3种根结线虫快速灵敏的鉴定。     

Genetic variation among field isolates of Pyrenopeziza brassicae

Amplified fragment length polymorphism (AFLP) markers were used to determine genetic diversity and population structure of Pyrenopeziza brassicae , the causal agent of light leaf spot of Brassica spp. Fungal isolates were sampled from six regions in the UK, one region in Germany and one region in France. A high level of genetic diversity was found ( H _T = 58%), with most variation attributed to within regions ( H _S = 43%), which suggests that sexual reproduction is frequent. F _ST values suggested significant population differentiation between England and the continent, but not between Scotland and England and Scotland and the continent. Overall, a moderate but significant level of regional differentiation was found ( F _ST = 16 ± 4.0). There was no correlation between F _ST values and distance, indicating that long-distance dispersal by natural factors does not occur at high frequencies. However, the lack of differentiation among populations from Aberdeen, Winchester and Cambridge suggests that seed transmission or other artificial methods of dispersal may be important.     

A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.     

58种中(草)药对植物寄生线虫Meloidogyne javanica和Pratylenchu svulnus的药效研究

 为寻找化学杀线虫的替代物,首次进行中国草药杀植物寄生线虫试验,从500多种有记载的杀线虫和昆虫的中草药中挑选58种进行筛选试验。研究表明,在试验条件下,22种中草药能杀死根结线虫Meloidogyne javanica,26种对短体线虫Pratylenchus vulnus有很强药效,其中9种明显杀死它们。试验还指出,大部分中草药都有不同程度的麻醉作用,受麻醉作用的线虫当转入水中后会复苏并恢复活力,麻醉作用程度随药物种类而异并随时间增加而明显加强。     

Genetic variation in Danish populations of Erysiphe graminis f.sp. hordei: estimation of gene diversity and effective population size using RFLP data

Genetic variation of the barley powdery mildew fungus ( Erysiphe graminis f.sp. hordei ) was estimated in three Danish local populations. Genetic variation was estimated from the variation amongst clones of Egh , and was therefore an estimate of the maximum genetic variation in the local populations. The average gene diversity, Ĥ , was estimated as 0.84. The effective population size was estimated as: log₁₀ ( N^ _e ) = 0.64 − log₁₀(μ), or 4.4 × 10⁹, assuming a nucleotide mutation rate ( μ) of 10⁻⁹ per base per generation. There was no significant genetic differentiation between locations.     

甘肃省白银市保护地蔬菜根结线虫病病原鉴定

2012年5月-7月在甘肃省白银市采集了番茄、黄瓜、豇豆等几种保护地蔬菜的25个根结线虫群体.直接解剖获得雌虫,通过雌虫会阴花纹观察,结合酯酶和苹果酸脱氢酶(MDH-EST)测定,鉴定出所收集的25个群体中有16个为南方根结线虫[Meloidogyne incognita (Kofoid&White)],9个为爪哇根结线虫[M.javanica (Treub)].南方根结线虫为优势种群,本次试验也是首次在甘肃省发现爪哇根结线虫.     

Inter-population variation in seed longevity for two invasive weeds: Chrysanthemoides monilifera ssp. monilifera (boneseed) and ssp. rotundata (bitou bush)

Seed longevity has a major influence on the success of weed management and eradication programmes. A correlation between responses to a controlled aging test performed at 45°C, 60% relative humidity (RH) and seed persistence in the field has recently been suggested. Here we investigated whether collections of differing quality of two closely related invasive weeds, Chrysanthemoides monilifera ssp. monilifera and ssp. rotundata (boneseed and bitou bush, respectively), had different seed longevity using the controlled aging test. Chrysanthemoides monilifera ssp. monilifera fruits were collected from across five Australian states, and C. monilifera ssp. rotundata from one state, covering their invasive ranges. Seed quality was assessed visually and using tetrazolium staining, and a series of germination tests established appropriate germination conditions. The controlled aging test was run for a subset of collections. Chrysanthemoides monilifera ssp. rotundata seeds died more quickly (time to lose 50% viability, P ₅₀ = 16 days) than the C. monilifera ssp. monilifera collections ( P ₅₀ = 47 days) when aged at 45°C and 60% RH. This difference was significant even considering the large differences in longevity between C. monilifera ssp. monilifera populations ( P ₅₀ = 35–61 days; probably due to differences in maturity at collection). Based on a published correlation, we predict that mature C. monilifera ssp. monilifera may have a long-lived (>3 years) seed bank and C. monilifera ssp. rotundata may have a transient (<1 year) seed bank. This suggests the two sub-species should be considered separately when designing effective control measures.     

中国南方稻田及其周边环境根结线虫种类鉴定

为了解根结线虫在中国南方稻区的分布和种类, 从15个省(直辖市/自治区)采集1 181份土样, 经生物测定发现312份土样中种植的水稻或番茄幼苗根系会产生根结, 阳性总检出率为26.42%。利用线虫特异性引物从171份阳性样品中鉴定出6种根结线虫:拟禾本科根结线虫Meloidogyne graminicola、北方根结线虫M.hapla、花生根结线虫M.arenaria、爪哇根结线虫M.javanica、象耳豆根结线虫M.enterolobii和南方根结线虫M.incognita, 分别占鉴定总样品数的88.89%、41.52%、29.82%、10.53%、4.09%和3.51%。被1、2、3、4、5、6种根结线虫混合侵染的土样比例分别为42.11%、41.52%、12.87%、2.92%、0.58%和0, 其中最常见的为拟禾本科根结线虫单独侵染(34.50%)、拟禾本科根结线虫与北方根结线虫(22.81%)或花生根结线虫(13.45%)混合侵染。本研究结果将为制定相应的传播阻断策略和防控措施提供参考。     

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.     

Protection of olive planting stocks against parasitism of root-knot nematodes by arbuscular mycorrhizal fungi

The effects were investigated, under controlled conditions, of single and joint inoculation of olive planting stocks cvs Arbequina and Picual with the arbuscular mycorrhizal fungi (AMF) Glomus intraradices , Glomus mosseae or Glomus viscosum , and the root-knot nematodes Meloidogyne incognita and Meloidogyne javanica , on plant performance and nematode infection. Establishment of the fungal symbiosis significantly increased growth of olive plants by 88·9% within a range of 11·9–214·0%, irrespective of olive cultivar, plant age and infection by M. incognita or M. javanica . In plants free from AMF, infection by Meloidogyne spp. significantly reduced the plant main stem diameter by 22·8–38·6%, irrespective of cultivar and plant age. Establishment of AMF in olive plants significantly reduced severity of root galling by 6·3–36·8% as well as reproduction of both Meloidogyne spp. by 11·8–35·7%, indicating a protective effect against parasitism by root-knot nematodes. Infection by the nematodes influenced root colonization by AMF, but the net effect depended on the AMF isolate–olive cultivar combination. It is concluded that prior inoculation of olive plants with AMF may contribute to improving the health status and vigour of cvs Arbequina and Picual planting stocks during nursery propagation.