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.     

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×10⁶ chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.     

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 Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

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.     

Fungi and actinomycetes associated with Meloidogyne spp. eggs and females in China and their biocontrol potential

A survey was conducted to determine the microflora on eggs and females of Meloidogyne spp. collected from plant roots and infested soil in China. A total of 455 fungal isolates belonging to 24 genera and 52 isolates of actinomycetes were obtained from 28 samples from greenhouses and fields in Hainan, Yunnan, Fujian, Hebei, Shandong, and Beijing. The predominant fungal species were Paecilomyces lilacinus (49.3% of the isolates), Fusarium spp. (7.9%), Pochonia chlamydosporia (6.9%), Penicillium spp. (5.7%), Aspergillus spp. (3.2%), and Acremonium spp. (2.8%). Actinomycetes were frequently encountered (10.3%) as well. A total of 350 isolates of nematophagous fungi and actinomycetes were evaluated for their parasitism of eggs and effects on egg hatch and juvenile mortality in vitro. Pathogenicity varied among isolates, and 29.1% of isolates parasitized over 90% eggs 4 days after inoculation. Results also show that seven isolates of fungi and actinomycetes reduced egg hatch rates to less than 10% contrasted to the control of 65.8%, and three isolates killed all hatched juveniles after 7 days. Seventeen fungal isolates and four actinomycete isolates with high pathogenicity in vitro were selected to test biocontrol efficacy in the greenhouse. They reduced tomato root gall index by 13.4-58.9% compared to the no treatment control.     

Effect of plant growth promoting rhizobacteria,nematode parasitic fungi and root-nodule bacterium on root-knot nematodes Meloidogyne javanica and growth of chickpea

Effects of plant growth promoting rhizobacteria (Pseudomonas putida MTCC No. 3604 and Pseudomonas alcaligenes MTCC No. 493) and parasitic fungi (Pochonia chlamydosporia KIA and Paecilomyces lilacinus KIA) were studied, alone and together with Rhizobium sp. (charcoal commercial culture) on the growth of chickpea and multiplication of Meloidogyne javanica. Individually, P. putida 3604, P. alcaligenes 493 and Rhizobium caused a significant increase in the growth of chickpea in both nematode inoculated and uninoculated plants. Inoculation of Rhizobium with a parasitic fungus or with plant growth promoting rhizobaterium caused a greater increase in the growth of plants inoculated with nematodes than caused by either of them singly. Individually, P. lilacinus KIA caused a greater increase in the growth of nematode inoculated plants than caused by P. putida 3604 or P. alcaligenes 493. P. lilacinus KIA caused a greater reduction in galling and nematode multiplication followed by P. chlamydosporia KIA, P. putida 3604 and P. alcaligenes 493. Combined use of P. lilacinus KIA with Rhizobium was better in reducing galling and nematode multiplication than any other treatment. P. putida 3604 caused a greater colonization of root than P. alcaligenes 493 while P. lilacinus KIA was isolated from more nematodes than P. chlamydosporia KIA.     

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 role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs

B-3 fungal isolate was isolated from soil samples of Gwangju in Korea. Based on morphological and phylogenetic analysis, it was designated as Lecanicillium antillanum B-3 (syn. Verticillium antillanum B-3). The fungus was a chitinolytic-nematophagous microorganism. B-3 chitinase activity from 0.5% swollen chitin broth medium reached the highest level on the sixth day and then plateaued until 12 days. B-3 isolate showed the high rate of parasitism on Meloidogyne incognita eggs with more than 90% infection rate on the third day after treatment. B-3 crude chitinase damaged the eggshell structures more than 78% based on lactoglycerol staining data at a final protein concentration of 14.6 µg mL^?1 on the fourth day following treatment. Partially purified chitinase with molecular 37 kDa from DEAE-Sephadex chromatography also showed damaging effect on the eggs. These results suggested that chitinase from B-3 isolate was responsible for degradation of M. incognita eggshell structures.     

Diversity and virulence of soil-dwelling fungi Isaria spp. and Paecilomyces spp. against Solenopsis invicta (Hymenoptera: Formicidae)

To comprehend the diversity and potential control of soil-dwelling fungi, Isaria and Paecilomyces, against the red imported fire ant Solenopsis invicta (Hymenoptera, Formicidae), an investigation was carried out between 2004 and 2008. From 258 soil samples collected from 16 central and southern provinces and cities in China, a total of 171 isolates of the genra Isaria and Paecilomyce were isolated, and the species I. javanicus, P. marquandii and I. fumosoroseus were found more abundant than I. cateniobliquus, P. carneus, P. inflatus and P. lilacinus. Geographic differences of isolating rates were observed as well. Samples from the southern areas had higher fungal isolating rates than those from the central areas. Subsequently, 47 isolates were further tested for pathogencity against the red imported fire ant. All isolates except P115 showed certain pathogenic potential (the mean is 52.3% at 4000 conidiospores/mL) to the ant. I. javanicus was the most effective species with a mean pathogenicity of 80.6%, while pathogenicities of P. marquandii, P. gunni and I. fumosoroseus were 44, 21 and 49%, respectively. Furthermore, the more effective isolates P028 of I. javanicus and P003 of I. fumosoroseus were tested in a virulence experiment. The LD₅₀ values of P028 and P003 against major and miner workers were determined as 412,280 and 854,451 conidiospores/cm², respectively. Meanwhile, the LT₅₀ values at 1000 conidiospores/cm² were 7.1 and 6.6 d in isolate P003 and 6.8 and 6.6 d in isolate P028.     

The potential of Isaria spp. as a bioinsecticide for the biological control of Nasutitermes corniger

The termite Nasutitermes corniger is a serious pest infesting urban areas of Brazil and many other countries. Control largely depends on synthetic pesticides whose indiscriminate use can impact the environment and the health of humans and other animals. Alternative strategies against insect pests, such as biological control by entomopathogenic fungi, could be effective while minimising these deleterious effects. We analysed the actions of the entomopathogenic fungi Isaria farinosa, Isaria fumosorosea, and Isaria javanica against the insect N. corniger. Our results indicated that the fungi examined were pathogenic against N. corniger, with I. farinosa ESALQ1355 being the most efficacious strain, resulting in the death of 95% of the workers (LC₅₀ 6.66?×?10⁴ conidia/mL) and 85% of the soldiers (LC₅₀ 6.81?×?10⁴ conidia/mL). This is the first report of the pathogenicity of Isaria spp. on N. corniger. These in vitro results suggest that I. farinosa ESALQ1355 demonstrates a significant biological potential for controlling N. corniger.     

喜树内生真菌的分离及其抗肿瘤活性代谢产物的筛选方法

从喜树(Camptotheca acuminata Decne.)的根、枝条、叶和果实中分离纯化了48株内生真菌,通过对各个菌株的少量发酵培养,HPLC分析结合色谱峰紫外扫描检测方法,以紫外扫描图谱的相似性为依据,对喜树内生真菌产生喜树碱结构类似物进行初步筛选,并进一步以抑瘤实验确证其抗肿瘤活性.结果证明,以该方法筛选到的10个内生菌株中有7个菌株发酵液对HL-60细胞增殖具有显著的抑制活性.相对于常规的生物活性筛选,高效液相色谱结合色谱峰紫外光谱的方法,在药用植物内生真菌活性次生代谢产物筛选研究中具有快速、高效的特点.     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

Performance of potential European biological control agents of Vincetoxicum spp. with notes on their distribution

Exploratory surveys were conducted in Europe to locate potential biological control agents of Vincetoxicum nigrum and Vincetoxicum rossicum and to collect known specialist herbivores of Vincetoxicum hirundinaria (Abrostola asclepiadis, Chrysolina a. asclepiadis, Eumolpus asclepiadeus and Euphranta connexa). Development of the most abundant herbivores found attacking leaves, roots, and developing seeds was evaluated in the laboratory on three Vincetoxicum spp. Field surveys revealed that the highest diversity of herbivores was associated with the abundant and widespread V. hirundinaria. No new herbivores were reported from V. hirundinaria or V. nigrum, but the leaf‐feeding noctuid Hypena opulenta was recorded for the first time attacking V. rossicum and V. scandens in Ukraine. Based upon larval survival and development and adult fecundity, the leaf feeders A. asclepiadis, C. a. asclepiadis, and H. opulenta, perform better on V. nigrum and V. rossicum than on V. hirundinaria. Larval performance of the root feeder E. asclepiadeus followed a similar pattern, but adult fecundity of this insect did not vary among host plant species. Immature development time of the pre‐dispersal seed feeder E. connexa is similar among hosts, but larvae grow larger on V. nigrum and V. hirundinaria than on V. rossicum. All herbivores are promising biological control agents of Vincetoxicum in North America and we have prioritized host range testing with H. opulenta and E. asclepiadeus.     

刺五加内生真菌分离鉴定及抗菌活性研究

为开发利用刺五加内生真菌资源,寻找新型抗菌物质,从刺五加根茎叶中分离纯化得到内生真菌71株,经菌落形态和显微形态去重后进行ITS序列分子鉴定为18个种,来自于4目、8科、11属,其中链格孢属与镰孢霉属为优势属。根、茎、叶中内生真菌多样性指数分别为1. 112 31、1. 523 658、0. 264 1。18种刺五加内生真菌中,16种内生真菌至少对一种指示菌有抗菌活性。CWJ-5(镰孢霉属Fusarium oxysporum)对金黄色葡萄球菌抑菌效果显著,抑菌直径27. 17±0. 07 mm; CWJ-6(链格孢属Alternaria alternata)对大肠埃希菌抑菌效果显著,抑菌直径18. 07±0. 01 mm。结果表明刺五加内生真菌资源丰富,有很大的开发利用空间,部分菌种抑菌活性显著,值得进一步研究。     

Occurrence of phytoalexins and phenolic compounds in endomycorrhizal interactions,and their potential role in biological control

This paper will review work mainly done during the last twenty years on the involvement of phytoalexin and phenolic compounds in mycorrhizal interactions. It has been observed that phytoalexins and associated molecules accumulate in roots after mycorrhizal infection, but less intensively and more slowly than in pathogenic interactions. Following mycorrhizal infection, enzymes of phenylpropanoid metabolism have been shown to be activated differentially. Some flavonoids and isoflavonoids have been reported to stimulate in vitro germination of mycorrhizal fungi or in vitro mycorrhizal infection, but their biological significance in signalling between the two symbiotic partners, and in biocontrol of plant disease by arbuscular mycorrhizal fungi, have not yet been elucidated.     

The biology of two Eretmocerus spp. (Haldeman) and three Encarsia spp. Forster and their potential as biological control agents of Bemisia tabaci biotype B in Australia

The performance, as measured by daily rate of parasitism and total parasitism, of five aphelinid species found in Australia parasitising Bemisia tabaci were compared on cotton, hibiscus, rockmelon, soybean and tomato. Two Eretmocerusspp., both indigenous to Australia, gave the highest levels of parasitism on each of the plant host species tested. The tritrophic interactions between B. tabaci, host plant species and Eret. mundus(Australian parthenogenetic form) (APF) were also examined. In general, more whiteflies were parasitised when cotton was the source host or rockmelon the test host. Parasitism was always low when tomato was either the source or test host. When parasitoids were transferred from rockmelon to cotton, parasitism declined. In contrast, parasitism increased when parasitoids were transferred from cotton to rockmelon. Parasitism also increased when parasitoids were transferred from soybean to rockmelon, yet failed to do so when shifted from soybean to cotton despite cotton normally being a better host. However, when parasitoids were transferred from cotton to soybean there was a marked increase in parasitism. Possible causes are discussed. The field cage trial demonstrated that parasitism by both Eretmocerus spp. increased with increasing whitefly density. Further, the increase in parasitism was not due to the presence of more parasitoids as neither the parasitoid-whitefly ratio nor the total number of parasitoids present had a significant effect on parasitism. The combination of the two species, gave similar levels of parasitism to that achieved by Eret. mundus(APF) alone. Subsequent identification of the emerged individuals indicated that over 50% of the parasitism was due to this species suggesting that it out-competed Eret. queenslandensis. Despite this competition, there was no evidence that overall control was compromised.     

Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish

AIMS: To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. METHODS AND RESULTS: Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. CONCLUSIONS: Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.     

Copper sorption and accumulation by the extraradical mycelium of different Glomus spp. (arbuscular mycorrhizal fungi) isolated from the same polluted soil

The form and localisation of Cu accumulation in the extraradical mycelium (ERM) of three arbuscular mycorrhizal fungi (AMF), isolated from the same polluted soil contaminated with the Cu and Arsenate, was studied. There were differences in the capacity of the ERM of the three AMF to sorb and accumulate Cu. Glomus caledonium BEG133 had a significantly lower Cu-sorption capacity than Glomus mosseae BEG132 and Glomus claroideum BEG134 isolated from the polluted soil as well as an isolate of G. mosseae BEG25 from a non-polluted soil. This was directly related to the cation exchange capacity (CEC) of the ERM of these fungi. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) linked to an energy dispersive X-ray spectrometer (EDAX) gave more detailed information, showing that the ERM of AMF from the polluted soil was able to accumulate Cu in the mucilaginous outer hyphal wall zone, cell wall and inside the hyphal cytoplasm. The EDAX spectra showed that the accumulated Cu was mainly associated with Fe in the mucilaginous outer hyphal wall zone and in the cell wall. Cu was associated with traces of arsenate inside the cytoplasm of the ERM of Glomus mosseae BEG132 but this was not visible inside the ERM of Glomus caledonium BEG133 or Glomus claroideum BEG134. This work suggests that the ERM of AMF is able to sorb and accumulate Cu, but different tolerance mechanisms exist between the three AMF isolated from the same polluted soil providing further evidence for functional diversity within populations of AMF in soils.