Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.     

铜绿假单胞菌生防菌株抑菌代谢产物及其生防应用研究进展

微生物次生代谢产物的研究对开发微生物源农药具有重要意义。近年来一系列根际来源的铜绿假单胞菌被分离和鉴定,因其产生抑菌次生代谢产物,具有很好的生物防治效果。本文将系统综述铜绿假单胞菌生防菌株的种类及其抑菌代谢产物的多样性,并进一步介绍铜绿假单胞菌生防菌株的抑菌代谢产物合成机制及其遗传改造,简要讨论铜绿假单胞菌生防菌株抑菌代谢产物在生物防治上的应用和前景。     

Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

Assessment of the biocontrol potential of a Trichoderma viride isolate: Part I: Establishment of field and fungal cellar trials

A field trial has been set up to assess the biological control potential of a Trichoderma viride isolate, T60. This isolate had been shown in previous laboratory tests to be particularly effective against certain basidiomycete decay fungi. Wood was treated with T60 spores using vacuum pressure impregnation in a pilot preservation plant. Treated stakes were planted in the field site along with CCA-treated and untreated control samples. Replicate samples were also set up in an accelerated decay facility employed to give a comparison to the field trial results. This paper describes the setting up and monitoring of the field and fungal cellar trials, and presents results of moisture monitoring and sapstain assessment which indicate that Trichoderma viride isolate T60 has a marked effect on the rate of sapstain development under certain conditions. The paper also discusses the efficacy of pressure impregnation of spore suspensions for use as biological control agents     

Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.     

Cotinus coggygria Scop.: An overview of its chemical constituents,pharmacological and toxicological potential

The Anacardiaceae Lindl. family comprises of many species which are used in nutrition and in traditional folk medicine for the treatment of several human diseases. Cotinus coggygria Scop. commonly known as “smoke tree”, is a commercial ornamental plant with high medicinal usages, belongs to the family Anacardiaceae. The present review provides a comprehensive report of empirical investigations on important pharmacological activities and phytochemical screening of essential oils and extracts. Relevant information was collected from scientific journals, books, and reports via library and electronic search using Medline, PubMed, Google Scholar, ScienceDirect, Web of Science, and Scopus. The plant has been extensively investigated in a broad range of studies to provide scientific evidence for folklore claims or to find new therapeutic uses. Numerous activities namely antioxidative, antibacterial, antifungal, antiviral, anticancer, antigenotoxic, hepatoprotective and anti-inflammatory have been demonstrated for all parts of these plants by in vivo and in vitro studies. Essential oils and extracts showed various pharmacological and biological properties which make them an effective remedy for various kinds of illnesses. Considering data from the literature, it could be demonstrated that C. coggygria possesses diverse bioactive properties and immense utilization in medicine, health care, cosmetics and as health supplements.     

Fungal pathogens of Euphorbia heterophylla and E. hirta in Brazil and their potential as weed biocontrol agents

A two-year survey of the fungi associated with two important congeneric pantropical weeds, Euphorbia heterophylla and E. hirta, was conducted in part of their native range in southern Brazil. Sampling was concentrated mainly in Rio de Janeiro State and ten species were identified as pathogens of these weeds. Two taxa, Botrytis ricini and Uromyces euphorbiae, were common to both weed hosts. Alternaria euphorbiicola, Bipolaris euphorbiae, Melampsora sp., Oidium sp. and Sphaceloma poinsettiae were recorded only from E. heterophylla, whereas Colletotrichum gloeosporioides, Sphaceloma sp. and Sphaerotheca fuliginea were restricted to E. hirta. Botrytis ricini and Colletotrichum gloeosporioides are new records for E. hirta, and Alternaria euphorbiicola and Sphaerotheca fuliginea are new host records for Brazil. Bipolaris euphorbiae, previously identified as Helminthosporium sp., is considered to be the correct name for the causal agent of a major disease of E. heterophyllum in Brazil. The potential of these pathogens as biocontrol agents is discussed and the mycobiota associated with both these weeds worldwide is reviewed.This revised version was published online in October 2005 with corrections to the Cover Date.     

The fungus Phoma multirostrata is a host-specific pathogen and a potential biocontrol agent for a broadleaf weed

Since the increasing prevalence of herbicide-resistant weeds and herbicide bans, the use of biological controls with mycoherbicides become an innovative approach of weed control. In this study, we verified the pathogenicity of Phoma multirostrata TBRC 12769 against the common weed in Thailand, tridax daisy (Tridax procumbens), with its mechanism of infection unveiled by fluorescence microscopy. P. multirostrata directly penetrated through epidermal cells, stomata, and trichomes at 48 h post-inoculation. The hyphae also propagated in the lumen of the trichome, enabling the fungus to grow subcuticular to neighboring weed tissues at the bases of leaf trichomes. The necrotic pattern emerged around the trichome. During necrosis, unicellular chlamydospores were also detected inside the leaf trichomes, suggesting an overwintering stage under stress and nutrient-depleting conditions. Trichomes of weed leaves were found to be key infection sites for pathogenesis. Topical application of conidial suspension on T. procumbens potted plants led to 60–98% and 65 and 87% disease incidence under laboratory and greenhouse conditions, respectively, on days 15–20 post-inoculation. The 16-h dew period incubation results in a sharp increase by 37% in the pathogenicity rate. The greenhouse trials verified that the fungus is non-pathogenic to eight crops. Our LC-MS analysis indicated that norharman, a known bioherbicidal compound, and other compounds were detected in the supernatant fraction of fungal culture, of which resulted in a blight symptom on T. procumbens leaves. This study demonstrated that the P. multirostrata isolate is an effective mycoherbicide for this broadleaf weed.     

Isolation and functional characteristics of the fungus Hypoxylon spp. Sj18 with biocontrol potential

A fungus with biocontrol potential was isolated from the roots of hickory trees. The strain named sj18 was classified as a member of the genus Hypoxylon (Hypoxylaceae) after multigene phylogenetic analysis (beta-tubulin gene, internal transcribed spacer, 28S large subunit ribosomal RNA gene, and RNA polymerase II subunit gene). The strain grew well on a PDA with an optimum temperature range between 32 and 34 °C. The fungus had obvious inhibitory effects on Botryosphaeria dothidea, Colletotrichum gloeosporioides, and Gibberella moniliformis in fumigation experiments on solid agar plates. In an inoculation experiment of Chinese cabbage, the fungus was also found to have an obvious repellent effect on cabbage caterpillars. In vitro experiments on Petri dishes showed that the fermentation broth of the sj18 strain could kill 100% of Bursaphelenchus xylophilus within 8 h even if the fermentation broth was diluted 8 times. The inoculation test of Arabidopsis thaliana showed that the fungus could promote the lateral root formation of plants and significantly increase their aboveground biomass. Through the analysis of solid phase microextraction (SPME), it was found that the main volatile components of the fermentation products were azulene 65.39% (61.77% + 3.62%), caryophyllene 7.41%, and eucalyptol 6.83% according to the peak area ratio. Therefore, sj18 can be used as a candidate for the further research and development of biocontrol agents.     

Rapid suppression of defence enzymes and compounds by sheath blight (Rhizoctonia solani) and sheath rot (Sarocladium oryzae) toxins in rice cell suspension cultures

To understand the suppression mechanisms against disease resistance in rice, we took advantage of the fact that suspension cultured cells exhibit many of the defence responses that are characteristic of intact tissues. In this study we constitutively measured the Rhizoctonia solani and Sarocladium oryzae toxins, induced and suppressed levels of phenylalanine ammonia lyase, peroxidase, superoxide dismutase, phenols, catalase, β-1,3-glucanase and chitinase in rice suspension cultured cells. The addition of Rhizoctonia solani and Sarocladium oryzae toxins separately in suspension cultured cells shows the suppression of defence enzymes and compounds at 24 h and 48 h respectively except SOD. The rice cultivar IR50 delays the disease suppression effect when compared to the other cultivars viz., Pusa Basmati and Co 43. The PR proteins (namely β-1,3-glucanase and chitinase) activities in rice suspension cultured cells were reduced during 48 h and 72 h after the addition of Rhizoctonia solani toxin, whereas the activities were suppressed only after 72 h when inoculated with Sarocladium oryzae toxin. Selective suppression of these defence enzymes and compounds by Rhizoctonia solani and Sarocladium oryzae toxin shows that toxins play a major role during pathogenesis in rice cells.     

Refining the biological factors affecting virulence of Botryosphaeriaceae on grapevines

Botryosphaeriaceae isolates of six species were assessed for their potential to infect grapevine tissues other than their tissues of isolation, primarily to determine sources of inocula that could contribute to bunch rot. Pathogenicity tests were conducted in vitro on berries and wood and in vivo on dormant buds of cultivars Chardonnay and Shiraz in glasshouse and field experiments. Tissue specificity and variation in virulence for different isolates was assessed. All isolates were able to infect and cause symptoms on detached 1‐year‐old canes and mature berries. Virulence was not affected by origin tissue and varied between isolates and within species. Inoculation of dormant buds did not affect bud burst or further development of shoots and fruit, however, a small number of Botryosphaeriaceae were reisolated from bunches at later growth stages. We conclude that Botryosphaeriaceae species are important pathogens of both the vegetative tissues and wood of grapevines. Grapevine wood infected with Botryosphaeriaceae could act as a source of inoculum for reproductive and vegetative tissue. Equally, the vegetative and reproductive tissues can also act as inoculum sources for wood infection. Therefore, all sources of inocula should be taken into consideration when developing management strategies for Botryosphaeria bunch rot and Botryosphaeria canker diseases.     

Taxonomy of parasitoid wasps in China: An overview

The parasitoid Hymenoptera are one of the most important groups of natural enemies of agriculture and forestry pests and have been used as biological control agents in IPM programs for a long time. The systematic research on parasitoids and their associations with hosts lays a solid base for biological control of pests. Here we summarize the taxonomic studies undertaken on the parasitoid wasps in China in the last eight decades. At present about 48 families in 12 superfamilies of parasitoid Hymenoptera are known in China, of which the majority of the families have been studied to some extent while a number of them are still poorly studied. Around 6000 species of 32 families which dealt with in this paper are known in China, and some of them have been successfully and widely used in the existing biological control while others are potentially useful in future biocontrol program. This overview provides the international audience a general idea about the current state of the systematic study of parasitoid Hymenoptera conducted in China, the known species of parasitoids and the native and introduced species used in biocontrol in China.     

侧孢短芽孢杆菌AMCC 100017的分离鉴定及其生防潜力

【目的】从土壤中分离、筛选和鉴定具有抑制病原真菌活性等生防效果的菌株,为进一步开发利用具有良好定殖能力的生防菌株奠定基础。【方法】利用对峙试验筛选拮抗菌并评价其拮抗性能,根据其形态特征、生理生化特性、16S r RNA基因序列测定及Gen Bank序列相似性分析进行分离菌株的分类鉴定,并通过福林酚法测定该菌株的蛋白酶活力。【结果】从山东泰安各种类型土壤中分离得到侧孢短芽孢杆菌(Brevibacillus laterosporus),保藏编号为AMCC100017。平板对峙试验结果表明该菌株对多种植物病原真菌均有较强的拮抗作用,尤其是对镰刀菌属致病菌拮抗效果明显。另外,本试验还初步验证该菌能产生较高活性的胞外蛋白酶。【结论】侧孢短芽孢杆菌AMCC 100017在作物真菌病害生物防治方面,有较好的开发和利用潜力,并可望应用于线虫生物防治。     

Application of actinomycetes as biocontrol agents in the management of onion bacterial rot diseases

This study was conducted to achieve biological control for the post-harvest onion bacterial rot diseases with the aid of Egyptian isolates of actinomycetes. In this respect, 45 actinomycetes strains were isolated from Egyptian soils and screened for their antagonistic effect against onion bacterial rot pathogens; Erwinia carotovora subsp. carotovora and Burkholderia cepacia. The most two active strains were identified based on their cultural, morphological and molecular properties as Streptomyces lavendulae HHFA1 and Streptomyces coelicolor HHFA2, the latter was most potent and so was used in vivo (pots and field) for controlling onion bacterial rot. S. coelicolor HHFA2 application resulted in enhancement in the photosynthetic pigments and some foliar growth parameters of onion plants confirming its growth promoting effect. The results of the post-harvest estimation of the disease incidence (DI) of the onion bacterial rot throughout storage revealed that, the application of S. coelicolor HHFA2 reduced the DI pronouncedly comparing with the untreated control and confirm its successful role in the biological control of onion bacterial rot diseases.     

Effect of the trap crop Solanum sisymbriifolium and two biocontrol fungi on reproduction of the potato cyst nematode,Globodera pallida

The potato cyst nematode, Globodera pallida, is one of the most important pests of potato worldwide. Owing to regulatory considerations and potential environmental impact, control options for this nematode are becoming increasingly limited. Solanum sisymbriifolium and biological control agents offer viable alternative options for controlling G. pallida. Therefore, experiments were conducted to determine the effect of the nematode trap crop S. sisymbriifolium, alone or in combination with the biocontrol agents Trichoderma harzianum or Plectosphaerella cucumerina, on population decline of G. pallida. Experiments were conducted for three different ‘cropping systems’: potato (Solanum tuberosum), S. sisymbriifolium, or soil only (fallow), each followed by a potato crop. Soil was amended with P. cucumerina, T. harzianum or left unamended, and then infested with nematodes at a rate of five eggs g^?1 of soil. After 16 weeks in the greenhouse, plants were removed and the soil containing cysts was refrigerated at 4°C for 8 weeks, and then planted to potato. Cysts of G. pallida were counted after an additional 16‐week period. The P_f/P_i of G. pallida was significantly reduced by 99% in potato following S. sisymbriifolium compared to both the potato‐following‐fallow and the potato‐following‐potato treatments. Amendment of soil with T. harzianum significantly reduced P_f/P_i of G. pallida by 42–47% in the potato‐following‐potato but not in either the potato‐after‐fallow nor in the potato‐after‐S. sisymbriifolium cycles which supports evidence that the plant species may play a role in the biocontrol activity of this fungus. Addition of the fungus P. cucumerina resulted in a 64% decrease in P_f/P_i in the potato‐following‐fallow in one experiment, and an 88% decrease in P_f/P_i in potato‐following‐potato but the decrease in P_f/P_i was not consistent over all experiments. However, both biocontrol fungi resulted in lower numbers of progeny cysts after an initial 16‐week incubation with potato. To look at the effect of varied population density of the nematode on efficacy of S. sisymbriifolium to reduce G. pallida populations, potato, S. sisymbriifolium, or barley were planted into soil infested with G. pallida at rates of 5, 20 or 40 eggs g^?1 soil applied as cysts (20, 80 or 160 cysts pot^?1). After 16 weeks, numbers of cysts produced in each treatment were determined for each infestation rate. No new cysts were recovered from either S. sisymbriifolium or barley treatments, confirming that neither plant is a host for G. pallida. High numbers of cysts were recovered with potato. Soil from each treatment (containing original cysts and newly‐formed cysts when present) were then planted with potato. After an additional 16 weeks, few cysts were found in the potato‐after‐ S. sisymbriifolium treatments regardless of initial infestation rate. When potato followed barley, numbers of cysts were similar to those found after a single cycle of potato, indicating that the barley crop had no effect on the survival of initial inoculum. Overall, these results suggest that S. sisymbriifolium has potential to significantly reduce G. pallida populations, and also that the cropping system (i.e. the sequence of non‐host and host plants) may play a significant role in the efficacy of fungal biological control agents.     

An overview on the natural enemies of Rhynchophorus palm weevils,with focus on R. ferrugineus

Rhynchophorus palm weevils are large insects belonging to the family Dryophthoridae. All Rhynchophorus species are polyphagous and have a similar life history but some are major pests because of the serious economic damage they cause, in particular to several species of the family Arecaceae. Here we review the natural enemies of Rhynchophorus species in both their native and introduced regions of the world, to assess the possibility of biological control of this taxon. Moreover, particular attention is paid to the well-studied and harmful species Rhynchophorus ferrugineus, about which more information is available, and to its natural enemies in the Mediterranean region, because the impact of this pest in this recently colonized area is particularly remarkable and also the recent trend in species management is looking for indigenous natural enemies.More than 50 natural enemies have been reported to attack Rhynchophorus species, even if most of them are associated to R. ferrugineus (Olivier), highlighting the lack of information on the other species of the genus. Pros and cons of all the biological control agents are then discussed: among the considered organisms, fungi are noteworthy to be considered for inclusion in integrated pest management programs.Overall, our overview underlines the need to increase knowledge on natural enemies of all the species of the genus Rhynchophorus, to isolate more virulent strains and to determine the optimum conditions for the actions of the biocontrol agents.     

Effects of temperature on the establishment potential in the U.K. of the non-native glasshouse biocontrol agent Macrolophus caliginosus

Abstract This study investigated the effect of temperature on the development and winter survival of the predatory mirid Macrolophus caliginosus Wagner, recently introduced into the U.K. as a biocontrol agent for glasshouse whitefly Trialeurodes vaporariorum . The developmental threshold for M. caliginosus calculated by three methods was between 7.3 and 8.4 °C, with a day-degree requirement per generation varying between 472 and 524 day-degrees. It was estimated that under outdoor conditions M. caliginosus could complete two generations per year in the U.K. All life stages of M. caliginosus had supercooling points around −20 °C, with some pre-freeze mortality evident in both acute and chronic low temperature exposures. Acclimation increased survival of nymphal M. caliginosus from approximately 24–52 days when exposed to a constant 0 °C. Provision of prey extended survival of nymphs in the laboratory at a constant 5 °C from 39 to 64 days and in the field by c . 150 days. The results are discussed in the context of the occurrence and establishment of M. caliginosus in the U.K. and the need to develop a reliable risk assessment system for non-native species used in glasshouse biocontrol.     

Effect of temperature on antagonistic and biocontrol potential of shape Trichoderma sp. on Sclerotium rolfsii

Sclerotium rolfsii is a destructive soil-borne and postharvest plant pathogen. Use of the antagonistic fungus Trichoderma sp. has been earlier reported by us to successfully control this pathogen under postharvest conditions. In the present paper we report on the effects of temperature on the growth and biocontrol potential of Trichoderma sp. on S. rolfsii. Experimental results indicated that S. rolfsii and Trichoderma sp. have different temperature optima for growth: 30–35 °C for the pathogen and 25–30 °C for the antagonist. In dual culture, Trichoderma overgrew S. rolfsii at 25 °C and 30 °C, but at 35 °C and 37 °C, S. rolfsii overgrew the colony of Trichoderma. Trichoderma produced higher concentration of fungitoxic metabolites in broth culture at higher temperatures. In bioassays using ginger slices and whole rhizomes, it has been demonstrated that Trichoderma is not very effective in suppressing S. rolfsii at temperatures above 30 °C. In light of these results, possible mechanisms of biocontrol of S. rolfsii as a postharvest pathogen has been discussed. Storage temperature has been suggested as a critical factor in biocontrol of S. rolfsii. This revised version was published online in June 2006 with corrections to the Cover Date.