Parasitism of Sedentary Stages of Heterodera glycines by Isolates of a Sterile Nematophagous Fungus

ABSTRACT Isolates of a sterile fungus designated ARF (Arkansas fungus) can be separated into two groups, ARF-C and ARF-L, that differ morphologically and in their ability to suppress numbers of Heterodera glycines on soybean. Our objectives were to determine if the two ARF groups differed in their ability to parasitize juveniles, females, and eggs in the rhizo-sphere of soybean and to proliferate in soil. The experiments were conducted in a greenhouse using soil infested with homogenized ARF mycelium. The ARF-L isolates parasitized more juveniles and young females than did the ARF-C isolates. Suppression of these stages was 67% for ARF-L and 12% for ARF-C isolates 14 days after nematode inoculation. When soybean plants containing gravid females were transplanted into fungus-infested soil, ARF-L isolates parasitized 55 to 98% of nematode eggs, whereas ARF-C isolates parasitized 0 to 22%. In both heat-treated and nonheated soil, the biomass of mycelial mats, a measure of relative proliferation, tended to be greater for ARF-L than for ARF-C isolates. The ability of ARF-L isolates to parasitize a large percentage of both pre-reproductive stages and eggs of H. glycines may contribute to its effectiveness as a biological control agent.     

Depth Distribution of Rotylenchulus reniformis Under Different Tillage and Crop Sequence Systems

ABSTRACT The population density of the reniform nematode, Rotylenchulus reniformis, was monitored at depths of 0 to 30, 30 to 60, 60 to 90, and 90 to 120 cm in a tillage and crop sequence trial in south Texas in 2000 and 2001. Main plots were subjected to three different tillage systems: conventional tillage (moldboard plowing and disking), ridge tillage, and no-tillage. Subplots were planted with three different crop sequences: spring cotton and fall corn every year; spring cotton and fall corn in one year, followed by corn for two years; and cotton followed by corn and then grain sorghum, one spring crop per year. The population density of R. reniformis on corn and grain sorghum was low throughout the soil profile. In plots planted with spring cotton and fall corn every year, fewer nematodes were found at depths of 60 to 120 cm in the no-tillage and ridge tillage systems than in the conventional tillage system. Population densities were lower at depths of 0 to 60 cm than at 60 to 120 cm. Soil moisture and cotton root length did not affect nematode population densities in the field. When soil was placed in pots and planted with cotton in the greenhouse, lower population densities developed in soil taken from depths of 0 to 60 cm than in soil from depths of 60 to 120 cm. Final nematode populations were similar in size in soil from the different tillage systems, but reproductive factors were higher in soil from plots with reduced-tillage systems than in soil from plots with conventional tillage. Reduced-tillage practices lowered the risk of increases in R. reniformis populations and reduced population densities following 2 years of non-hosts throughout soil depths, but population densities resurged to the same high levels as in soil planted with cotton every year during one season of cotton.     

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.     

Isolation, Characterization, and Distribution of a Biocontrol Fungus from Cysts of Heterodera glycines

ABSTRACT Seventy-six populations of Heterodera glycines were collected from 33 counties in 10 states of the United States along the Mississippi and Missouri Rivers in 1992 and 1993. A sterile hyphomycete fungus of an unnamed taxon, designated ARF18 and shown to be a parasite of eggs of H. glycines, was isolated from eggs and cysts of 10 of the populations from Kentucky, Louisiana, Mississippi, and Tennessee. Ten isolates of ARF18 obtained in this study and seven isolates obtained in earlier studies were characterized for cultural morphology on several growth media, the ability to produce sclerotium-like structures (SLS) on cornmeal agar, growth rates, pathogenicity to eggs of H. glycines in vitro, and mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs). All 17 isolates of ARF18 readily grew on potato dextrose agar, cornmeal agar, and nutrient agar. Based on colony morphology and SLS appearance on cornmeal agar, the isolates could be grouped into two morphological phenotypes. Isolates that produced SLS that were composed of a compact mass of hyphae were designated ARF18-C, whereas isolates that produced SLS composed of a mass of loosely clumped hyphae were designated ARF18-L. Only minor differences in growth rates were detected among the ARF18-C and ARF18-L isolates. All 10 ARF18-C isolates, which were from Arkansas, Louisiana, Mississippi, and Tennessee, belonged to a single mtDNA RFLP haplotype. The seven ARF18-L isolates shared many comigrating mtDNA restriction fragments with one another, but belonged to three distinct mtDNA RFLP haplotypes. Ability to infect eggs of H. glycines in vitro varied considerably among the various isolates of ARF18. In particular, several of the ARF18-C isolates were consistently able to infect over 50% (mean = 70.0%, standard deviation = 16%) of the eggs of H. glycines, whereas ARF18-L infected eggs to a lesser degree (mean = 25%, standard deviation = 27%). ARF18-C was isolated only from H. glycines populations from below 37 degrees N latitude. The presence of ARF18 was associated with soils with Mg levels <314 kg/ha, cyst numbers >4.5 per 100 cm(3), and iron levels >203.5 kg/ha; or with Mg levels >314 kg/ha and Na levels <121 kg/ha. The widespread distribution of ARF18 and the ability of some isolates to aggressively colonize eggs of H. glycines are indications that it has potential as a biological control agent for H. glycines.     

Investigation of alternatives to methyl bromide for management of Meloidogyne javanica on greenhouse grown tomato

The root galling index and the densities of eggs in roots and juveniles in soil of the root-knot nematode Meloidogyne javanica (Treub) Chitwood on tomato, and the effect of these on crop yield were assessed in greenhouse experiments applying various treatments at two different sites in Crete, Greece. Tomato crops were grown for four cycles by rotating nematode-resistant (first and third spring crops) with susceptible (second and fourth autumn crops) cultivars and receiving the following treatments: (a) untreated control; (b) methyl bromide application before the first and third crops; (c) application of the fungus Pochonia chlamydosporia (Goddard) Zare, Gams & Evans before planting the first, third and fourth crops with a supplementary application three weeks after the beginning of the fourth crop; (d) application of oxamyl in both sites and fenamiphos in site 1 only at the second and fourth crops; (e) combination of treatments (c) and (d). The fungus density in soil was monitored three weeks after application and at the end of each crop, when roots were lifted. Pochonia chlamydosporia had a variable establishment and did not control the nematode. Its pathogenicity on eggs was not demonstrated, as in all cases galls were big, with all egg masses inside and protected from infection. The methyl bromide treatment significantly reduced root galling and egg production compared to other treatments in all crop cycles and the yield of the fourth crop was significantly greater. Nematicides reduced nematode densities compared with untreated controls and the fungus treatment, but they were less effective than methyl bromide and resulted in increased yield in one site only.     

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina

ABSTRACT To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.     

安徽省首次发现肾形肾状线虫危害瓜蒌

肾形肾状线虫Rotylenchulus reniformis Linford & Oliveira是作物根部半内寄生植物线虫, 被列为危害最严重的十大植物线虫之一。本文对安徽省新发生瓜蒌肾形线虫病的病原进行鉴定并对其生活史进行初步研究。利用形态学和分子生物学将病原线虫鉴定为肾形肾状线虫R.reniformis, 系统发育分析表明其属于A型。肾形肾状线虫在瓜蒌根部完成生活史需要24 d, 每条雌虫平均产卵64粒, 繁殖系数R=(11.12±0.62)。本文系首次报道肾形肾状线虫在安徽省发生危害, 瓜蒌为肾形肾状线虫的新寄主。本研究为进一步掌握肾形肾状线虫的分布区域及潜在寄主及综合防控提供了理论依据。     

Influence of Temperature and Inoculum Density of Fusarium oxysporum f. sp. ciceris on Suppression of Fusarium Wilt of Chickpea by Rhizosphere Bacteria

The effects of temperature and inoculum density of Fusarium oxysporum f. sp. ciceris race 5 on suppression of Fusarium wilt in chickpea (Cicer arietinum) cv. PV 61 by seed and soil treatments with rhizobacteria isolated from the chickpea rhizosphere were studied in a model system. Disease development over a range of temperatures (20, 25, and 30 degrees C) and inoculum densities (25 to 1,000 chlamydospores per gram of soil) was described by the Gompertz model. The Gompertz relative rate of disease progress and final amount of disease increased exponentially and monomolecularly, respectively, with increasing inoculum densities. Disease development was greater at 25 degrees C compared with 20 and 30 degrees C. At 20 and 30 degrees C, disease development was greater at 250 to 1,000 chlamydospores per gram of soil compared with 25 to 100 chlamydospores per gram of soil. At 25 degrees C, increasing inoculum densities of the pathogen did not influence disease. Nineteen Bacillus, Paenibacillus, Pseudomonas, and Stenotrophomonas spp. out of 23 bacterial isolates tested inhibited F. oxysporum f. sp. ciceris in vitro. Pseudomonas fluorescens RGAF 19 and RG 26, which did not inhibit the pathogen, showed the greatest Fusarium wilt suppression. Disease was suppressed only at 20 or 30 degrees C and at inoculum densities below 250 chlamydospores per gram of soil. Bacterial treatments increased the time to initial symptoms, reduced the Gompertz relative rate of disease progress, and reduced the overall amount of disease developed.     

Parasitism of macroconidia, chlamydospores and hyphae of Fusarium culmorum by mycoparasitic Pythium species

Parasitism of macroconidia and endoconidial chlamydospores of Fusarium culmorum by Pythium oligandrum was studied on water agar (WA), corn-meal agar (CMA) and glass slides. Loss of cytoplasmic content in F. culmorum spores was followed by complete degradation, and P. oligandrum produced an abundance of oogonia on the parasitized macroconidia. A simple method for assessing the relative aggressiveness of isolates is presented, based on the percentage of macroconidial cells devoid of cytoplasm. Parasitism of macroconidia by P. acanthophoron , P. oligandrum and P. periplocum , but not by the plant pathogenic species, P. tracheiphilum , was demonstrated by this method. Interactions between hyphae of P. oligandrum and F. culmorum on WA resulted in an increase in the number of oogonia of P. oligandrum and a decrease in the sporulation of F. culmorum . The ability of isolates of P. oligandrum , P. periplocum , P. acanthophoron and P. mycoparasiticum to suppress disease symptoms caused by F. culmorum on barley seedlings was demonstrated in a greenhouse test.     

Nematophagous Verticillium spp. in soils infested with Meloidogyne spp. in Cuba: Isolation and screening

Root-knot nematodes cause substantial economic loss of yield in coffee plantations and vegetable crops in Cuba. At present, methods to control the nematodes are ineffective or inappropriate and alternatives are being sought. The nematophagous fungus Verticillium chlamydosporium (Goddard) was isolated from soils collected from coffee plantations and infected root-knot nematode eggs from roots of tomato plants grown in these soils. A total of 83 isolates were collected and identified morphologically as V. chlamydosporium var. chlamydosporium, V. chlamydosporium var. catenulatum, V. psalliotae, V. suchlasporium and an isolate of V. chlamydosporium var. chlamydosporium with unusually large dictyochlamydospores. From these, 24 that represented a range of origins were selected and screened for their ability to parasitize eggs of root-knot nematodes, colonize the rhizosphere of barley roots and produce chlamydospores. None of the isolates grew at temperatures below 15°C and V. suchlasporium grew at a faster rate at lower temperatures than the other isolates. These were also screened in the glasshouse and V. chlamydosporium var. catenulatum caused the greatest reduction in nematode populations. One isolate of each subspecies of V. chlamydosporium was tested with the standard, Rothamsted isolate 10, on a range of host plants. The greatest reduction in numbers of nematodes occurred on tomato plants (cv. Pixie). The Rothamsted isolate 10 reduced numbers of nematodes toa greater extent than the other isolates, and therefore has the greatest potential as a biological control agent of root-knot nematodes.     

Application of Trichoderma and Gliocladium in alginate pellets for control of Rhizoctonia damping-off

Alginate pellets were prepared from wet fermentor biomass of 11 isolates of Trichoderma spp. and Gliocladium virens , with wheat bran as a food base carrier. Pellets with eight of the isolates reduced survival (34-78%) of Rhizoctonia solani in infested beet seed in soil. Pellets containing a T. harzianum isolate (Th-58) and a T. hamatum isolate (TRI-4) were the most effective. All isolates significantly reduced growth of the pathogen from infested beet seed into natural soil. Populations of isolates proliferated in soil to 10⁶−10¹¹ colony-forming units/g (cfu) from propagules within the pellets. Pellets with TRI-4 reduced pathogen survival and growth (>70%) in six different soils and were effective against six R. solani isolates in a natural loamy sand. Survival of R. solani in infested beet seed was not reduced when TRI-4 pellets were added to soil 1-6 weeks before the pathogen; however, saprophytic growth was prevented. Small amounts of biomass (3.0–7.5 g wet weight) in pellets were as effective as a large amount (300 g) in suppressing the pathogen. The storage of pellets for more than 6 weeks at 5 or 25C reduced their effectiveness against R. solani. Pellets prepared with four and three of the 11 isolates prevented damping-off of cotton and sugar beet in the greenhouse, respectively.     

Pathozone dynamics of Meloidogyne incognita in the rhizosphere of tomato plants in the presence and absence of the nematophagous fungus, Pochonia chlamydosporia

Pathozone dynamics were derived for approx. 50 Meloidogyne incognita juveniles infecting a single root of tomato under controlled conditions from a point source of inoculum and described using simple, non-linear models. The pathozone decayed sigmoidally with distance, but increased over time as progressively more nematodes were able to infect the root. Despite the reported ability of M. incognita juveniles to travel up to 50 cm in some conditions, the maximum width of the pathozone for a single tomato root was estimated as 18·1 mm. It is conjectured that this was because of (i) diffusion from a point source of inoculum, (ii) a small infection court (a single root tip) and (iii) the limited life span of the nematode. A second experiment was used to assess the effect of the nematophagous fungus Pochonia chlamydosporia on the pathozone dynamics of the nematode. This fungus is known to produce nematicidal products in vitro , which may affect invasion of roots by the free-living nematode. To examine the possibility of a change in the position of the site of infection, changes in the probability of gall formation along the root length were also examined. In the absence of P. chlamydosporia , the pathozone dynamics of M. incognita were very similar to those of the first experiment. It was shown that P. chlamydosporia did not significantly affect the pathozone dynamics of M. incognita nor the site of gall formation, which appear to have little importance for the role of the fungus as a biological control agent.     

离体培养玉米螟赤眼蜂的初步研究

本文首次报导用人工饲料离体培养 玉米螟赤眼蜂从卵到成虫。饲料组成为柞蚕蛹血淋巴或柞蚕蛹整体匀浆液、蛋黄和牛奶。用此饲料作卵浆制成的人工卵,按1粒卵接6头蜂的比例接蜂,寄生率达90％以上。每一被寄生后出蜂的卵可得成蜂34—37头,其中雌性90％、展翅率95％,出蜂卵率56—63％。文中还讨论了赤眼蜂在人工卵内数最少不能化蛹的原因。     

Vegetative Compatibility Groupings of Verticillium dahliae from Cotton in Mainland China

One hundred and fourteen isolates of Verticillium dahliae obtained from cotton and eggplant in mainland China were successfully assigned to two vegetative compatibility groups (VCGs) except for one self-incompatible isolate. Eleven isolates were strongly compatible with T9, the tester strain of the cotton defoliating pathotype, forming a linear growth of wild type with abundant microsclerotia and dense mycelia between compatible nitrate-nonutilizing mutants. The remaining 102 isolates were grouped into the non-defoliating VCG2, although the strength of the reaction varied; some isolates were strongly compatible with the tester strain while others were only slightly compatible. All VCG1 isolates including T9 showed the same defoliating symptom in greenhouse inoculation tests. This study confirmed the presence of the defoliating pathotype (VCG1) of V. dahliae in mainland China.     

四川省棉田寄生线虫种类及肾形线虫与棉花枯萎病发生关系的研究

 1988~1990年,用浅盘法对四川省10个县市的棉花枯萎病圃和连作重病地土壤进行线虫的分离和鉴定,共检出植物寄生线虫7个属和5个种。用肾形线虫(Rotylenchulus renifor-mis)和枯萎菌(Fusarium oxysporum f.sp.vasinfectum)混合接种,棉田枯萎病发病率和严重度比单接枯萎病菌显著提高,且病情增长更快,土壤中肾形线虫的数量也增多。     

拟粗壮螺旋线虫对棉花的致病力及其与棉枯萎病的复合症

 温室测定结果:拟粗壮螺旋线虫(Helicotylenchus pseudorobusius)接种量在每100cm³土壤100~1000条的条件下,接种30天后棉苗生长量比无线虫对照苗显著降低(P<0.05)。抗枯萎品种86-1在单接枯萎菌无线虫处理中不发生枯萎病,在枯萎菌(Fusarium oxysporum f. sp. vasinfectum)接种量为每克土7.5×10⁵孢子和线虫(每100cm³±500~1000条)组合处理中发病,棉苗枯萎病情指数与线虫接种量之间呈正相关(γ=0.97)。田间试验结果表明:防治线虫和枯萎病复合症用薰蒸剂棉隆每m²8~14g处理土壤比用非薰蒸性杀线虫剂克百威(呋哺丹),丙线磷(益收宝)和克线磷的效果更好。     

Characterization of AG-13, a Newly Reported Anastomosis Group of Rhizoctonia solani

ABSTRACT Rhizoctonia solani anastomosis group (AG)-13 was collected from diseased roots of field grown cotton plants in Georgia in the United States. Isolates of AG-13 did not anastomose with tester isolates of AG-1 through AG-12. Mycelium of all isolates of AG-13 were light brown but darkened as cultures aged. All isolates produced aerial mycelium. Concentric rings were visible after 3 to 4 days of growth but disappeared as cultures aged and darkened. Individual sclerotia were up to 1.5 mm in diameter, similar in color to the mycelium, and generally embedded in the agar. Clumps of sclerotia up to 5 mm in diameter were produced on the agar surface. All attempts to induce basidiospore production were unsuccessful. The 5.8S region of the rDNA from isolates of AG-13 was identical in length and sequence to isolates of all other AGs of R. solani. Length and sequence of the internal transcribed spacer (ITS) regions of rDNA from isolates of AG-13 were unique among AGs of R. solani. Similarity between AG-13 and other AGs of R. solani ranged from 68 to 85% for ITS region 1 and 85 to 95% for ITS region 2. Selected isolates of AG-13 caused minor or no damage to barley, cauliflower, cotton, lettuce, potato, and radish in laboratory or greenhouse studies.     

Efficacy of dose-dependent indigenous microbial insecticides against cotton aphid,Aphis gossypii Glover (Homoptera: Aphididae) at various temperature regimes

Strain selection based on temperature may be warranted when choosing an isolate for development as a microbial control agent. To this end, the effects of three temperature regimes, namely 20, 25 and 30°C, on the virulence of four Beauveria and two Metarhizium isolates against the cotton aphid, Aphis gossypii, were investigated under controlled conditions, 65–70% relative humidity, and a photoperiod of 12:12 (light: dark) hours. The isolates did vary significantly in their activities of efficacy over a range of temperatures. The highest mortality and lowest survival times were observed at 25°C for DLCO41 and at 30°C for DLCO87; while mortality decreased and survival time increased at temperatures of 20°C. Besides the tested fungal isolate DLCO87 had the lowest LC₅₀ value (6.84 × 10⁵ conidia mL^?1) at 30°C. The promising result of this study should enable us to conduct further studies to determine the potential use of the fungus as an agent against Aphis gossypii both in greenhouse and under field conditions.     

云木香内生细菌的分离鉴定及其对大丽轮枝菌的抑制作用

从药用植物云木香中分离得到41株内生细菌, 采用细菌16S rRNA基因测序的方法对这些菌株进行鉴定, 结果显示, 这些云木香内生细菌分别属于6个属, 其中假单胞菌属Pseudomonas和拉恩氏菌属Rahnella为优势菌属。采用对峙培养方法对这些菌株进行筛选, 发现其中12株内生细菌对大丽轮枝菌Verticillium dahliae具有抑制作用, 其中有2株属于拉恩氏菌属Rahnella, 7株属于假单胞菌属Pseudomonas, 3株属于沙雷氏菌属Serratia。12株内生细菌对棉花黄萎病的温室防效评估结果表明, 假单胞菌属菌株R1-6、R1-13和S1-2对棉花黄萎病的温室防效达到55%以上, 具有潜在的生产利用价值。     

肾形肾状线虫（Rotylenchulus reniformis）浙江杭州 群体形态学和分子特性及寄主范围

 肾形肾状线虫（Rotylenchulus reniformis）是一种重要的蔬菜线虫,寄主范围和分布很广。本文根据形态学特征和形态测量将来自浙江省杭州市蔬菜上的肾状线虫群体鉴定为肾形肾状线虫,为浙江省首次报道。该群体的形态学特征及形态测量值与模式标本基本吻合,个别形态测量值(比值)有变异。通过对该肾形肾状线虫群体的rDNA中ITS区和编码28S RNA基因中的D2D3区的PCR扩增和序列测定,明确二序列长度分别为808 bp和786 bp;序列分析发现该群体ITS序列与肾形肾状线虫种内一些两性生殖群体的亲缘关系较近,同源性高达99.5%～100%,而与一孤雌生殖群体同源性为92.4%;编码28S RNA基因中的D2D3区与已报道群体的同源性达到99.4%,相似度较高;本研究还发现杭州肾形肾状线虫群体的寄主涉及11个科16个属的果蔬。