Persistence of the nematophagous fungus Paecilomyces lilacinus strain 251 in soil under controlled conditions

The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study.     

Purification and characterization of Paecilomyces lilacinus dextranase

Two dextranase isoenzymes [endo-(1,6)-α-d-glucan-6-glucanohydrolase, EC 3.2.1.11] have been isolated from a crude enzyme powder prepared from the culture supernatant of Paecilomyces lilacinus. Purification was achieved by means of a two-stage ion-exchange chromatography on DEAE-cellulose. Dextranase I was recovered with a 35.3-fold increase in specific activity and a yield of 16%; dextranase II was purified 19-fold with a yield of 4%. The characteristics of the isoenzymes were very similar; both exhibited maximum hydrolytic activity at pH 4.5 and 55°C. Activation energies for thermal inactivation were 402 and 330 kJ mol^?1 for dextranase I and II, respectively. The dextranases were not inhibited by EDTA or N-ethylmaleimide.     

淡紫拟青霉(Paecilomyces lilacinus)培养条件的优化

以查氏培养基为基础,在碳源、氮源、pH值、温度等单因子条件研究的基础上,采用正交实验对淡紫拟青霉的培养条件进行了优化研究,得到了适合其生长的发酵培养基配方。实验结果表明,以蔗糖为碳源(60g/L),硝酸铵为氮源(1.5g/L),pH5～6,培养温度为30℃时最适合该菌的生长。     

根癌农杆菌介导的增强型绿色荧光蛋白基因在淡紫拟青霉中的转化

为了实现增强型绿色荧光蛋白基因 (egfp) 在生防真菌淡紫拟青霉9410菌株中的转化,借助中间质粒pcDNA3.1(-) 构建nptⅡ-egfp融合基因的表达载体pUPNGT,然后采用根癌农杆菌介导的转化法将egfp基因转化到淡紫拟青霉9410菌株中。PCR检测和Southern blotting分析结果表明,egfp基因以单拷贝形式整合到淡紫拟青霉9410的基因组中。荧光显微镜观察结果显示,转化子在488 nm下能产生绿色荧光。这些结果说明egfp基因已成功转化至淡紫拟青霉9410菌株并获得表达。这些工作可为淡紫拟青霉在不同条件下的防效评价、环境安全评价等提供新的途径和方法。     

淡紫拟青霉胞外多糖的分离、纯化及结构分析

淡紫拟青霉NH-PL-03菌株的胞外多糖粗提物对枯萎病病原菌-尖孢镰刀菌具有较好的抑制效果,文中对淡紫拟青霉胞外多糖进行了分离纯化和结构分析,以期为其构效关系研究奠定基础。采用乙醇沉淀法从淡紫拟青霉发酵液中提取粗多糖,经Sevage法脱蛋白后,过Superdex-G75凝胶层析柱分离得到胞外多糖EP-1。紫外分光法和Sephacryl S-200 HR凝胶层析柱检测EP-1为均一多糖,Sephacryl S-200柱层析测得EP-1的分子量为35.2 kDa,完全酸水解后纸层析检测EP-1的单糖组成中仅有葡萄糖,红外光谱、高碘酸氧化和Smith降解结果表明EP-1的化学结构是以β-(1,3)糖苷键连接而成的无分枝的葡聚糖。刚果红络合试验表明EP-1在稀的碱溶液中以3股螺旋构象存在。     

摇床转速对淡紫拟青霉菌生长的影响

设置不同摇床转速来调节通气量,以淡紫拟青霉菌株NHPL03培养过程的OD值、pH值、菌丝重量、孢子量、毒力为指标,研究通气量对淡紫拟青霉菌NHPL03生长的影响。结果表明:淡紫拟青霉菌可在多种摇床转速(通气量)条件下生长。通气量不仅影响菌丝生长与孢子生成而且影响毒力产物分泌,菌丝生长与孢子生成的最佳摇床转速为100r/min,培养8d菌丝干重达到最高值1.10g/50mL,产孢量达28.5×106个/mL。从毒力强度看,低转速条件60r/min总体毒力水平最高,培养8d菌液对茎线虫校正致死率达到47.1%。     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

食线虫真菌是植物寄生线虫的重要天敌,它们所产生的胞外水解酶(蛋白酶、几丁质酶和胶原蛋白酶等)能够降解线虫体壁和卵壳中的蛋白质及几丁质等结构成分并在侵染过程中发挥着重要的作用。本文中,我们发现刀孢轮枝菌Lecanicillium psalliotae对南方根结线虫Meloidogyne incognita卵具有较强的侵染能力。为了进一步研究刀孢轮枝菌胞外几丁质酶的性质,我们通过简并引物设计和DNA walking方法从刀孢轮枝菌的基因组中成功地克隆得到一个内切几丁质酶基因Lpchi1,该几丁质酶编码基因含有3个内含子,编码423个氨基酸。同源性和系统发育分析表明,不同生防真菌来源的几丁质酶具有较高的同源性并根据分子量的大小形成三个不同的进化分枝。     

Histology of the interactions of Paecilomyces lilacinus with Meloidogyne incognita on Eclipta alba (L.)

Abstract

Under greenhouse conditions, the experiment was conducted to test the efficacy of opportunistic and nematophagous hyphomycete, Paecilomyces lilacinus against root-knot nematode, Meloidogyne incognita on Eclipta alba. The treatment comprised inoculation of E. alba with M. incognita alone (N) and in combination with P. lilacinus (one week before (T₁), simultaneously (T₂), one week after (T₃), two weeks after (T₄) and three weeks after (T₅)). The results showed that the application of P. lilacinus before one week of nematode inoculation (T_l) was more effective than other treatments. A significant enhancement was noticed in the growth and yield of E. alba. Root-knot and egg mass indices were suppressed due to destruction of the mature females and the egg masses. Histopathological studies revealed the presence of P. lilacinus hyphae in and around the females and the eggs.     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

Nematophagous fungi, one of the natural enemies of nematodes, have been employed in biological control. Extracellular enzymes secreted from nematophagous fungi, including protease, chitinase and collagenase serve as virulence factors of infection. In this study, we found Lecanicillium psalliotae can penetrate the eggs of the root-knot nematode Meloidogyne incognita and influence development of the eggs. A chitinase gene Lpchi1 was isolated from L. psalliotae using degenerate primers and DNA-walking technique. Comparison of the chitinase amino acid sequences from different pathogenic fungi revealed that the enzymes were highly similar. The phylogenetic analysis demonstrated that the chitinases derived from different fungi were clustered into three main clades corresponding to different molecular weight.     

蓖麻提取物和淡紫拟青霉对南方根结线虫的防治作用

通过杀线活性测定及盆栽试验,研究了蓖麻提取物和淡紫拟青霉(Paecilomyces lilacinus)对南方根结线虫(Meloidogyne incognita)的杀线活性及防治效果.结果表明:蓖麻碱不影响淡紫拟青霉孢子的萌发.蓖麻碱和淡紫拟青霉均具有较强杀线活性,蓖麻碱处理对南方根结线虫的卵孵化抑制率和二龄幼虫死亡率分别达61.7%和59.2%,显著高于对照处理;蓖麻碱和孢子液复合处理接种南方根结线虫的番茄苗后,植株平均根结数为15±3,显著低于对照的平均根结数37±2,株高、鲜重和根长增长率分别比对照提高38.5%、44.0%和57.0%.说明蓖麻提取物和淡紫拟青霉能减轻线虫危害,对番茄南方根结线虫病控制效果明显.     

Efficacy of Pseudomonas fluorescens and Paecilomyces lilacinus against Meloidogyne graminicola infecting rice under system of rice intensification

The talc-based formulations of the plant growth promoting rhizobacterium, Pseudomonas fluorescens and egg parasitic fungi, Paecilomyces lilacinus, were evaluated as seed treatment, soil application and combination of both for the management of M. graminicola in fields of rice grown under system of rice intensification. Both the bioformulations significantly reduced the root invasion and soil populations of M. graminicola but P. fluorescens was most effective when applied as seed cum soil application and seed treatment alone. Effect of these treatments was comparable with the standard chemical carbofuran application. The introduced P. fluorescens survived significantly in rice roots when applied as seed cum soil application and seed application alone than as soil application. There was significant increase in phenol, peroxidase and chitinase accumulation in plants treated with P. fluorescens. Application of bioagents had positive influence on growth parameters such as plant height, root length, shoot weight, root weight and number of tillers per hill. Application of P. fluorescens as seed cum soil treatment resulted in higher grain yield, which was 20.6%–26.9% increase over control followed by P. fluorescens as seed treatment alone that increased grain yield of rice by 10.7%–11.2% than control. However, economic returns per investment was higher when P. fluorescens was applied as seed treatment alone (1:8.8–1:12.0 incremental cost benefit ratio) followed by the P. fluorescens as seed cum soil treatment (1:6.2–1:9.7 incremental cost benefit ratio).     

Enzymatic analysis and in vitro ovicidal effect of Pochonia chlamydosporia and Paecilomyces lilacinus on Oxyuris equi eggs of horses

The aim of this work was to analyse the enzymatic activity and in vitro ovicidal effect of Pochonia chlamydosporia (VC1 and VC4) and Paecilomyces lilacinus (PL1) on Oxyuris equi eggs of horses. The growth of isolates and their enzymatic production were evaluated on agar media supplemented with gelatin (GA), casein (CA), olive oil (OOA) or starch (SSA). The ovicidal effect was evaluated according to morphological alterations. Following, the P. chlamydosporia crude extract production and proteolytic content was evaluated (VC1 and VC4) in liquid medium at the interval of 15, 30, 45 and 60 min during incubation of P. chlamydosporia and P. lilacinus which grew and showed enzymatic activity on agar media (GA, CA, OOA and SSA). At the 15th day, VC1, VC4 and PL1 showed results on GA, CA, OOA and SSA media, for Type 3 effect of (78, 54, 52 and 68%), (72, 50, 58 and 78%) and (62, 57, 65 and 63%). Pochonia chlamydosporia was able to grow in liquid medium (gelatin) and at Day 5 showed proteolytic activity. The results of the present work suggest that P. chlamydosporia and P. lilacinus can be used in the biological control of O. equi eggs.     

Effect of storage conditions on the survival of two potential biocontrol agents of nematodes,the fungi Paecilomyces lilacinus and Pochonia chlamydosporia

The nematophagous fungi Paecilomyces lilacinus and Pochonia chlamydosporia have been extensively studied as biological control agents for plant-parasitic nematodes. This study describes the formulation of alginate pellets containing mycelia of these fungi and also describes the effect of storage conditions on shelf-life of the pellets. The shelf-lives of P. lilacinus and P. chlamydosporia, which were measured monthly for 6 months, were significantly improved at low temperatures and low water activity (a _w) values (<0.33). Vacuum did not affect the viability of the formulated P. lilacinus but increased the viability of P. chlamydosporia. Carbon dioxide reduced the activity of P. lilacinus as compared to ambient air but increased the activity of P. chlamydosporia. Nitrogen, however, significantly improved the viability of both fungi. The optimal parameters of each factor for our formulation of P. lilacinus and P. chlamydosporia included a temperature range of 4 to ?20°C, a _w=0.12, and a nitrogen-filled atmosphere.     

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels

The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.     

Effects of a Commercial Formulation of Paecilomyces lilacinus Strain 251 on Overseeded Bermudagrass Infested with Belonolaimus longicaudatus

Belonolaimus longicaudatus is an important parasite of both warm-season bermudagrass and winter overseed grasses used on golf courses in the southeastern United States. Field trials were conducted to study the effects of a commercial formulation of Paecilomyces lilacinus strain 251 applied to overseed grasses during the winter and early spring on population density of B. longicaudatus and bermudagrass health in late spring after bermudagrass broke dormancy. These studies found that P. lilacinus reduced numbers of B. longicaudatus in most cases, but not below damaging levels. Multiple applications of 1 × 10¹⁰ spores/m² were generally more effective than 2 × 10¹⁰ spores/m² in reducing nematode numbers and improving turf roots. These results indicate that application of this formulation of P. lilacinus strain 251 to overseeded turf in the spring may be a useful integrated pest management tool for B. longicaudatus on bermudagrass, but is not sufficient as a stand-alone nematode management tactic.     

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 an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromycota: Hyphomycetes) obtained from Formosan subterranean termites (Isop., Rhinotermitidae)

Abstract:  An isolate of Paecilomyces fumosoroseus was obtained from Coptotermes formosanus collected in Hong Kong, and a commercially available isolate of Metarhizium anisopliae , were both tested against C. formosanus shipped live from China. Survivorship of termites treated with a suspension of 5 × 10⁵ M. anisopliae conidia/ml and kept alone declined more rapidly than for those treated at the same concentration of P. fumosoroseus conidia. At a 5 × 10⁶ conidia/ml concentration, no significant differences in terms of termite survivorship were observed between the two fungal species. However, among termites kept in groups of 10 after treatment, those sprayed with P. fumosoroseus conidia at either 5 × 10⁵ or 5 × 10⁶ conidia/ml had significantly lower survivorship than those sprayed with M. anisopliae conidia. All the cadavers of termites treated with P. fumosoroseus and kept alone sporulated and among grouped termites 29% of the cadavers sporulated. By comparison, 53% of the cadavers of termites treated with M. anisopliae and kept alone sporulated, and only 4% of the cadavers of treated termites kept in groups sporulated.     

Effect of temperature on growth, germination, germ-tube extension and survival of Paecilomyces lilacinus strain 251

Paecilomyces lilacinus and in particular the commercial strain 251 has been intensively tested for biological control of plant parasitic nematodes. Since this species has been mentioned in a number of reports concerning infection of humans, the human health risk for Paecilomyces lilacinus strain 251 was investigated. The effects of time, temperature and growth medium on radial colony growth and germination were determined. Additionally, exposure to 36°C and its effect on germ-tube extension and on survival of conidia was evaluated. Radial growth was significantly affected by temperature, growth medium and their interaction. Optimum temperatures were between 24 and 30°C, but no growth was found at 36°C. Germination rate was significantly influenced by time, medium, temperature and their interactions. The optimum temperature range for germination was between 28 and 30°C. Formulated conidia were capable of germinating at 36°C. However, studies on germ-tube extension conducted at 36°C showed a delay in development for 28-49 h and no further germ-tube extension was found after exposure for 80-95 h. Slopes of survival curves were significantly influenced by the type of conidia tested. In general, conidia did not survive exposure to 36°C for 168 h. These experiments indicate the temperature conditions where the strain is likely to be active and provide supporting data for full environmental and health risk assessments of biocontrol fungi.     

Functional expression in Beauveria bassiana of a chitinase gene from Ophiocordyceps unilateralis,an ant-pathogenic fungus

A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter.