Understanding the movement of root-knot nematodes encumbered with or without Pasteuria penetrans

Pasteuria penetrans is a naturally occurring bacterial parasite of plant parasitic nematodes showing satisfactory results in a biocontrol strategy of root-knot nematodes (Meloidogyne spp.). The endospores attach to the outside nematode body wall (cuticle) of the infective stage second-stage juveniles (J2) of Meloidogyne populations. Optimal attachment level should be around 5–10 endospores per juvenile, as enough endospores will initiate infection without reducing the ability of the nematode to invade roots. Greater than 15 endospores may disable the nematode in its movements, and invasion may not take place. In this research, evidence is provided that P. penetrans spores disturbed the nematode forward movement by disorganising the nematode's head turns. The results based on Markov chain and Cochran probability model show that even a low number of 5–8 spores of P. penetrans attached to the nematode cuticle have a significant impact on that movement, which plays a role in nematode locomotion.     

Suppression of Meloidogyne hapla populations by Hirsutella minnesotensis

The effects of the endoparasitic fungus Hirsutella minnesotensis on populations of Meloidogyne hapla from Michigan (MI), Rhode Island (RI), Connecticut (CT), Lyndonville, New York (NYL), Geneva, New York (NYG), and Wisconsin (WI) were studied in the greenhouse. Twenty-day-old tomato (cv. Rutgers) seedlings were inoculated with either 0 or 600 eggs of each nematode population mixed with either 0, 0.02, or 0.1 g of fresh H. minnesotensis mycelium 0.1 L^-1 of soil in pots containing 0.5 L of soil, and maintained at 25±2°C for 2 months. No effect of the fungal treatments and nematode treatments on tomato plant heights and shoot dry weights was observed. While all M. hapla populations were suppressed by H. minnesotensis, the degree to which each population was affected varied slightly. Across fungal treatments and nematode populations, the fungus reduced total number of nematodes in roots by 61-98%, with the highest for NYG and RI, intermediate for NYL and CT, and lowest for MI and WI populations. The study demonstrated that H. minnesotensis may be used as a potential suppressor of M. hapla in vegetable production systems in the Great Lakes Region.     

AMF和DSE组合菌剂促生防线虫病效应

本试验旨在探究丛枝菌根真菌（AMF）和暗隔内生真菌（DSE）组合菌剂对南方根结线虫Meloidogyne incognita发育、侵染、黄瓜根结线虫病以及黄瓜生长发育的影响,为进一步探索AMF和DSE协同发挥生理生态效应的作用机制奠定基础。试验对“津优35号”黄瓜接种南方根结线虫、Phoma leveillei（DSE）、Funneliformis mosseae（AMF）、Glomus versiforme（AMF）、Acaulospora laevis（AMF）和/或Scutellospora aurigloba（AMF）。结果表明,供试AMF与DSE 能促进黄瓜生长、提高产量和增强抗病性。AMF+DSE组合处理的菌根和DSE的定殖数量、株高、茎粗、地上部和根系干重、单株产量等显著优于单接种AMF或单接种DSE处理。以F. mosseae + P. leveillei组合抑制南方根结线虫的发育、降低线虫繁殖数量、根内定殖数量、发病率和根结指数的效果最好。     

Effects of Hirsutella minnesotensis and N-Viro Soil® on populations of Meloidogyne hapla

This study was conducted to determine the effects of Hirsutella minnesotensis (Hm), an endoparasitic fungus, and N-Viro Soil® (NVS), a recycled municipal biosolid, on Meloidogyne hapla greenhouse populations from Rhode Island (RI), Connecticut (CT), Geneva, New York (NYG), Lyndonville, New York (NYL), and Michigan (MI). In a greenhouse experiment, tomato (cv. Rutgers) seedlings were inoculated with 0 or 600 eggs of each nematode population and exposed to Hm mycelium (0 or 0.1 g fresh) and NVS (0 or 1 g 0.1 L^-1 of soil) in a factorial design. Hirsutella minnesotensis reduced nematode densities by 31-83% across nematode populations in one test, but only slightly reduced densities of NYG and CT populations in another test. NVS reduced nematode densities by 33-92% across populations in two repeated tests. The combination of the two agents resulted in greater nematode reduction compared with Hm alone, but not compared with NVS alone. Across all Hm and NVS treatments, reduction of nematode densities were generally greater in NYG, CT, and RI than in MI and NYL populations. This study demonstrated that Hm and NVS may be used to suppress different M. hapla populations.     

不同基质中番茄菌根苗的培育及其抗南方根结线虫的效果

根结线虫病是番茄主要的土传病害,特别是保护地长期种植会加重病害的发生。接种丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）能提高植物抗根结线虫的能力,促进植株生长发育,减轻病害。本试验旨在评价常用基质配合施用一种生物有机肥对番茄苗生长和菌根定殖的影响,以获得菌根发育良好且生长健壮的菌根苗,并通过盆栽实验验证菌根苗移栽于大棚连作土壤中对南方根结线虫的防治效果。结果表明,栽培于商业基质的番茄苗的株高、茎粗、地上部及根系干重和壮苗指数均显著高于草炭蛭石混合基质的番茄苗;草炭蛭石混合基质中施加生物有机肥能促进番茄苗的生长,但是显著抑制根内根孢囊霉Rhizophagus intraradices的侵染。草炭蛭石混合基质在培养过程中施加适量Hoagland营养液使番茄苗生长发育较好且根内根孢囊霉R. intraradices侵染率达到71%;商业基质培养的番茄苗壮苗指数更高,但侵染率较低（44%）。在草炭蛭石混合基质中接种变形球囊霉Glomus versiforme和根内根孢囊霉R. intraradices及其混合菌种培育的番茄苗对南方根结线虫Meloidogyne incognita的侵染和繁殖均表现出一定的抑制作用。按照单位重量根系进行统计,混合菌种显著降低了根结线虫的根结和卵块数量（分别降低了22.8%和23.5%）。本研究结果表明番茄菌根苗在草炭蛭石混合基质中AMF发育状况优于常用商业基质,在菌根应用中可优先选择;相对于单一接种AMF,混合接种的菌根化苗对南方根结线虫病害的抑制作用更好。     

Induced resistance by the mutualistic endophyte, Fusarium oxysporum strain 162, toward Meloidogyne incognita on tomato

The non-pathogenic endophytic fungus, Fusarium oxysporum strain 162, originally isolated from the endorhiza of tomato roots, reduces damage caused by Meloidogyne incognita, by inhibiting juvenile penetration of and development in the root. However, little is known about the mode of action of this endophyte fungus against the nematode. This study aimed at investigating how the endophyte affects nematode motility and survival and if induced resistance plays a role in the relationship. In a previous study, F. oxysporum strain 162 decreased nematode penetration of tomato up to 60%. In experiments using a split-root chamber to test for induced resistance, nematode penetration, number of galls, and number of egg masses were investigated 2 and 5 weeks after nematode inoculation. Split-root plants treated with F. oxysporum strain 162 showed 26-45% less nematode penetration, 21-36% less galls and a 22-26% reduction in the number of egg masses in the roots not directly inoculated with the fungus when compared to untreated control plants in repeated tests. In conclusion, inoculation of tomato plants with the non-pathogenic fungal endophyte F. oxysporum strain 162 resulted in a signficant reduction of nematode infection, which was in part due to induced resistance in the first 2-3 weeks after fungal inoculation.     

Interactions of Paecilomyces lilacinus strain 251 with the mycorrhizal fungus Glomus intraradices: Implications for Meloidogyne incognita control on tomato

The interactions of Paecilomyces lilacinus strain 251 with the arbuscular mycorrhizal fungus Glomus intraradices and their significance for the control of Meloidogyne incognita on tomato were investigated in greenhouse experiments. Application of P. lilacinus had no effect on the frequency and intensity of tomato root colonization by G. intraradices. Likewise, the decline of the nematophagous fungus densities after single application in soil was not affected by the presence of the mycorrhizal fungus. Single application of P. lilacinus, as pre-planting soil treatment, resulted in significant reduction of nematode damage. In contrast, mycorrhizal inoculation did not provide sufficient biocontrol. Combined application of the two agents did not enhance root protection compared to single treatments. Double treatment of mycorrhized seedlings with P. lilacinus, as seedling drench and pre-planting soil treatment, 4 and 1 week before transplanting, respectively, resulted in the highest reduction of the nematode damage. These results indicate the potential of the commercial P. lilacinus strain 251 and mycorrhiza for integration in nematode control strategies.     

Evaluation of a Native and Introduced Isolate of Pochonia chlamydosporia against Meloidogyne javanica

Growth chamber and plastic tunnel experiments were conducted to compare the ability of a native and introduced isolate of Pochonia chlamydosporia to colonize the rhizosphere of selected plant species and survive in soil. Effects of the isolates on population density of Meloidogyne javanica and yield of tomato after single or multiple fungal applications were also determined. In growth chamber experiments, both isolates showed a similar ability to colonise the rhizosphere of selected vegetables, except for the introduced isolate, which produced more colony forming units cm^-2 of root surface on tomato and cabbage than the native one. In the tunnel house, both isolates parasitized eggs of M. javanica, and the native but not the introduced isolate increased parasitism after multiple applications. The native isolate was recovered more frequently from soil, and was a better colonizer of tomato roots than the introduced one irrespective of the number of fungal applications. Multiple fungal applications of either isolate reduced the nematode gall rating, and the native isolate also reduced the final egg population in roots. Neither isolates reduced final nematode densities in soil or affected tomato yield when compared to untreated plots.     

The Feeding Behavior of Adult Root-knot Nematodes (Meloidogyne incognita) in Rose Balsam and Tomato

Meloidogyne incognita is a parasitic root-knot nematode that causes considerable yield loss in a wide range of plants. In this study we documented the movement of adult female nematodes for more than 2 hr in micro-slices of infected tomato (Solanum lycopersicum) and rose balsam (Impatiens balsamina) plants using light and video microscopy. Stylet thrusting was followed by short pumping actions of the esophagus, dorsal esophageal gland ampulla, and metacorpal bulb. Regular thrusting was normally accompanied by head turning and always preceded continuous stylet thrusting aimed at a single point (for 20 to 90 sec). Females often held the stylet in a protruded position, while pulsating the metacorpus bulb, for about 30 sec. Subsequently, the stylet was paused in a retracted position for 5 to 40 sec. This sequence of behavior took 290 to 380 sec to complete. The procedure developed in this study provides a useful cytological technique to investigate the interaction between root-knot nematodes and the giant cells formed by infected plants. Scanning electron microscopy revealed that the head of the adult nematode was located in the narrow intercellular spaces among several giant cells. The anterior part of the head of the adult was folded like a concertina, whereas that of the second-stage juvenile was not. The labial disc and medial lips of second-stage juveniles seemed expanded and sturdy, whereas those of the adult were star-shaped, appeared to be contracted, and softer. These morphological differences in the heads of adult and second-stage juveniles are discussed with respect to their movement.     

In-vitro Assays of Meloidogyne incognita and Heterodera glycines for Detection of Nematode-antagonistic Fungal Compounds

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.     

地上-地下植食性天敌对入侵植物空心莲子草与本地种莲子草种间关系的影响

生物入侵是全球生物多样性的主要威胁,外来种与本地种的种间竞争能力会影响其能否成功入侵。本研究选用入侵植物空心莲子草和其本地同属种莲子草为对象,探究其专食性天敌莲草直胸跳甲与南方根结线虫对空心莲子草与莲子草的生长及种间关系的影响。结果表明: 与无天敌胁迫相比,线虫处理显著降低了莲子草的株高(28.1%),但显著增加了空心莲子草的株高(52.8%)和莲子草的地上生物量(63.7%);跳甲处理显著降低了莲子草的株高(40.7%),对空心莲子草无显著影响;而跳甲与线虫的共同胁迫显著降低了莲子草的株高(35.3%)和空心莲子草的地下生物量(62.2%),显著增加了莲子草的地上生物量(69.1%);天敌胁迫对两种植物的茎粗、分枝数和根长均无显著影响。无天敌作用下,两种植物的相对邻体效应指数(RNE)均为正值,且空心莲子草的RNE比莲子草高21.3%;天敌胁迫下,空心莲子草的RNE均为负值,而莲子草的RNE在线虫或跳甲单独胁迫下为正值,在线虫和跳甲共同胁迫下为负值。表明地上-地下天敌互作可以使两种植物的种间关系发生改变,并可能促进空心莲子草的入侵。     

A New Root-Knot Nematode Parasitizing Sea Rocket from Spanish Mediterranean Coastal Dunes: Meloidogyne dunensis n. sp. (Nematoda: Meloidogynidae)

High infection rates of European sea rocket feeder roots by an unknown root-knot nematode were found in a coastal dune soil at Cullera (Valencia) in central eastern Spain. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European sea rocket plants and in artificially inoculated tomato (cv. Roma) and chickpea (cv. UC 27) plants. The species is herein described and illustrated and named as Meloidogyne dunensis n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by: (i) perineal pattern rounded-oval, formed of numerous fine dorsal and ventral cuticle striae and ridges, lateral fields clearly visible; (ii) female excretory pore at the level of stylet knobs, EP/ST ratio 1.6; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anteriorly to excretory pore and long, narrow, tapering tail; and (iv) males with lateral fields composed of four incisures anteriorly and posteriorly, while six distinct incisures are observed for large part at mid-body. Phylogenetic trees derived from distance and maximum parsimony analyses based on 18S, ITS1–5.8S-ITS2 and D2-D3 of 28S rDNA showed that M. dunensis n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. duytsi, M. maritima, M. mayaguensis and M. minor.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.     

Histopathology of Root-knot Nematode (Meloidogyne incognita) Infection on White Yam (Dioscorea rotundata) Tubers

White yam tissues naturally and artificially infected with root-knot nematodes were fixed, sectioned, and examined with a microscope. Infective second-stage juveniles of Meloidogyne incognita penetrated and moved intercellularly within the tuber. Feeding sites were always in the ground tissue layer where the vascular tissues are distributed in the tubers. Giant cells were always associated with xylem tissue. They were thin walled with dense cytoplasm and multinucleated. The nuclei of the giant cells were only half the size of those found in roots of infected tomato plants. Normal nematode growth and development followed giant cell formation. Females deposited eggs into a gelatinous egg mass within the tuber, and a necrotic ring formed around the female after eggs had been produced. Second-stage juveniles hatched, migrated, and re-infected other areas of the tuber. No males were observed from the tuber.     

Meloidogyne lusitanica n. sp. (Nematoda: Meloidogynidae), a Root-knot Nematode Parasitizing Olive Tree (Olea europaea L.)

A root-knot nematode from Portugal, Meloidogyne lusitanica n. sp., is described and illustrated from specimens obtained from olive trees (Olea europaea L.). Females of the new species have a characteristic perineal pattern with medium to high trapezoidal dorsal arch with distinct punctuations in the tail terminus area. The excretory pore is located posterior to the stylet, about 1.5-2.5 stylet lengths from the anterior end. The stylet is 17.1 μm long with pear-shaped knobs. Males have a rounded, posteriorly sloping head cap and head region not annulated. The robust stylet, 24.5 μ long, has large, elongate knobs. Mean length of the second-stage juveniles is 449.5 μm, stylet length 14.2 μm, and tail length 44.1 μm. Scanning electron microscope observations provide further details of perineal patterns and head and stylet morphology of females, males, and second-stage juveniles. Meloidogyne lusitanica n. sp. did not reproduce on any of the differential hosts used to separate the four most common Meloidogyne species. The common name "olive root-knot nematode" is proposed for M. lusitanica n. sp.     

Description of a Unique,Complex Feeding Socket Caused by the Putative Primitive Root-Knot Nematode,Meloidogyne kikuyensis

Meloidogyne kikuyensis produces unique galls that form on one side of the root resembling nitrogen-fixing nodules that are produced on legumes in response to infection by Rhizobium and related bacteria. The gall caused by this root-knot nematode is made up of a complex feeding socket composed of several giant cells that are ramified with xylem vessels extending perpendicular from the vascular cylinder. The anterior portion of the second-stage juvenile, which develops into an adult, plugs into this unique feeding socket. The socket and the surrounding parenchyma together form a gall that is very different in morphology from those typically caused by other species of root-knot nematodes. Even though M. kikuyensis was considered to be a primitive species because of its low chromosome count, the complexity of its feeding site and minor plant damage suggests a more derived systematic position.     

Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

Interaction between a fungal endophyte and root herbivores of Ammophila arenaria

The effect of an endophytic fungus (Acremonium strictum) on plant-growth related parameters of marram grass (Ammophila arenaria), and its potential as a protective agent against root herbivores (Pratylenchus dunensis and Pratylenchus penetrans, root-lesion nematodes) was investigated in two inoculation experiments under different conditions. Acremonium strictum-inoculated plants showed increased plant development in terms of root biomass in the first experiment and increased number of tillers in the second experiment and biomass was less suppressed by nematodes than the Acremonium strictum-free plants. In neither experiment did Acremonium strictum reduce multiplication of root herbivores. On the contrary, Acremonium strictum-inoculated plants seemed to increase herbivore multiplication. Plants infected with P. penetrans benefitted more from the endophytic fungus than those with P. dunensis in terms of total biomass. The effect of Acremonium strictum on interspecific competition was also analyzed by plant inoculation with both nematode species. In Acremonium strictum-free plants with mixed nematode inoculum, the total number of nematodes, compared to numbers observed in one-species inoculation, was less than expected, suggesting that interspecific competition took place. In Acremonium strictum-inoculated plants no interspecific competition was observed. Plants inoculated with P. dunensis, P. penetrans and Acremonium strictum showed decreased total biomass compared to Acremonium strictum-free plants inoculated with the same nematodes. The implications of increased tillering and root growth of plants with Acremonium endophytes are discussed in relation to the sand stabilizing role of Ammophila arenaria in coastal dunes.     

基于多基因分析对我国红曲霉属Monascus真菌的系统发育研究

红曲霉属Monascus具有重要的经济和生态价值,但对该属的物种识别和系统发育学研究有限。本研究首先对红曲霉属红色组内物种开展ITS和LSU序列的测定并综合分析,结合GenBank中相关物种序列及菌株的形态学特征,探讨红曲霉属的系统发育关系,鉴定出红色组3个种,弗罗里达组7个种。其次采用红曲霉色素表型控制基因簇部分基因pksKS序列进行分析,以期解决ITS和LSU等基因序列分析无法有效区分红色红曲与紫色红曲不同形态种的问题。通过分子克隆测序与直接测序结果的比较,以及对3个血红红曲菌株单核苷酸多态性的分析,解析了血红红曲种内遗传差异,首次在红曲霉属真菌中发现疑似红色红曲血红自然杂交种。最后综合ITS、LSU、pksKS序列和形态学分析的结果,统一了红曲霉属内物种的系统发育关系,确认红色组包括3个种：红色红曲M. ruber、紫色红曲M. purpureus、血红红曲M. sanguineus;发现3个变种：红色红曲发白变种M. ruber var. albidulus、紫色红曲橙色变种M. purpureus var. aurantiacus及紫色红曲火红色变种M. purpureus var. rutilus;弗罗里达组包括7个种：弗罗里达红曲M. floridanus、苍白红曲M. pallens、新月红曲M. lunisporas、阿根廷红曲M. argentinensis、累西腓红曲M. recifensis、黄色红曲M. flavipigmentosum、蜂蜜红曲M. mellicola。另外,发现1个疑似杂交种：红色红曲血红杂种M. ruber × sanguineus。结果表明,红曲霉属具有丰富的物种多样性,而且通过多基因和形态学分析可以将相近种区分开来。     

DNA repair in reduced genome: the Mycoplasma model

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species.