Combined use of biocontrol agents to manage plant diseases in theory and practice

Effective use of biological control agents (BCAs) is a potentially important component of sustainable agriculture. Recently, there has been an increasing interest among researchers in using combinations of BCAs to exploit potential synergistic effects among them. The methodology for investigating such synergistic effects was reviewed first and published results were then assessed for available evidence for synergy. Correct formulation of hypotheses based on the theoretical definition of independence (Bliss independence or Loewe additivity) and the subsequent and statistical testing for the independence-synergistic-antagonistic interactions have rarely been carried out thus far in studies on biocontrol of plant diseases. Thus, caution must be taken when interpreting reported "synergistic" effects without assessing the original publications. Recent theoretical modeling work suggested that disease suppression from combined use of two BCAs was, in general, very similar to that achieved by the more efficacious one, indicating no synergistic but more likely antagonistic interactions. Only in 2% of the total 465 published treatments was there evidence for synergistic effects among BCAs. In the majority of the cases, antagonistic interactions among BCAs were indicated. Thus, both theoretical and experimental studies suggest that, in combined use of BCAs, antagonistic interactions among BCAs are more likely to occur than synergistic interactions. Several research strategies, including formulation of synergy hypotheses in relation to biocontrol mechanisms, are outlined to exploit microbial mixtures for uses in biocontrol of plant diseases.     

A numerical study of combined use of two biocontrol agents with different biocontrol mechanisms in controlling foliar pathogens

Effective use of biocontrol agents is an important component of sustainable agriculture. A previous numerical study of a generic model showed that biocontrol efficacy was greatest for a single biocontrol agent (BCA) combining competition with mycoparasitism or antibiosis. This study uses the same mathematical model to investigate whether the biocontrol efficacy of combined use of two BCAs with different biocontrol mechanisms is greater than that of a single BCA with either or both of the two mechanisms, assuming that two BCAs occupy the same host tissue as the pathogen. Within the parameter values considered, a BCA with two biocontrol mechanisms always outperformed the combined use of two BCAs with a single but different biocontrol mechanism. Similarly, combined use of two BCAs with a single but different biocontrol mechanism is shown to be far less effective than that of a single BCA with both mechanisms. Disease suppression from combined use of two BCAs was very similar to that achieved by the more efficacious one. As expected, a higher BCA introduction rate led to increased disease suppression. Incorporation of interactions between two BCAs did not greatly affect the disease dynamics except when a mycoparasitic and, to a lesser extent, an antibiotic-producing BCA was involved. Increasing the competitiveness of a mycoparasitic BCA over a BCA whose biocontrol mechanism is either competition or antibiosis may lead to improved biocontrol initially and reduced fluctuations in disease dynamics. The present study suggests that, under the model assumptions, combined use of two BCAs with different biocontrol mechanisms in most cases only results in control efficacies similar to using the more efficacious one alone. These predictions are consistent with published experimental results, suggesting that combined use of BCAs should not be recommended without clear understanding of their main biocontrol mechanisms and relative competitiveness, and experimental evaluation.     

Regulation and use of biological control agents in Hungary

A wide range of biological control agents (BCAs) have been authorized and used in Hungary for the control of pests (including pathogens). BCAs are key elements of both integrated pest management and organic farming for different crops. Authorization of BCAs depends on the type of ‘active substance’. Micro‐organisms can be authorized under the EU Regulation (EC) No. 1107/2009 as plant protection products (PPP), while macro‐organisms are not within its scope. Plant protection tools (traps, mating disruption) are registered at a national level in Hungary, although mass traps and mating disruption pheromones are considered to be PPP. Plant extracts can be authorized as PPP, but some of them can be approved as a ‘basic substance’ and in this case they do not need further authorization. In Hungary, the vast majority of macro‐organisms used as BCAs have been mainly used in greenhouses and polytunnels for the control of thrips, whiteflies, aphids, leaf mining flies, phytophagous mites and lepidopteran pests in vegetable and ornamental crops. Recently a non‐indigenous chalcid wasp, Torymus sinensis, has been successfully introduced for the control of the oriental chestnut gall wasp, Dryocosmus kuriphilus, on chestnut (Castanea sativa) on different sites.     

Biosuppression of Botrytis cinerea in grapes

There is increasing interest in the use of biological control agents (BCAs) and plant resistance stimulants to suppress botrytis bunch rot in grapes, caused by Botrytis cinerea . Numerous different filamentous fungi, bacteria and yeasts have been selected as potential BCAs for control of grey mould based upon demonstrated antagonism towards B. cinerea. Biological suppression of the pathogen arises via competition for nutrients and space, the production of inhibitory metabolites and/or parasitism. Preformed and inducible grapevine defence mechanisms also contribute to disease suppression by preventing or delaying pathogenic infection. Furthermore, various biotic and abiotic agents can stimulate grapevine defence mechanisms and so elevate resistance to B. cinerea infection. Biosuppression of B. cinerea in vineyards, using BCAs and resistance stimulants, has been inconsistent when compared with that observed in controlled glasshouse or laboratory conditions. This may be attributable, in part, to the innate variability of the field environment. Research to improve field efficacy has focused on formulation improvement, the use of BCA mixtures and combinational approaches involving BCAs and plant resistance stimulants with complementary modes of action.     

木霉几丁质酶及其对植物病原真菌的拮抗作用

 木霉(Trichoderma spp.)作为重要的植病生防因子(biocontrol agents)一直受到普遍关注。木霉菌株产生的包括几丁质酶在内的细胞壁降解酶,在木霉重寄生中起重要作用。本文论述木霉几丁质酶的种类、诱导产生、理化特性及其对植物病原真菌的拮抗作用,并对木霉几丁质酶及其基因的生防应用进行讨论。     

木霉在植物病害生物防治中的应用及作用机制

木霉是植物病害生物防治中应用和研究非常广泛的一类生防真菌。本文阐述了木霉在多种植物病害防治上的应用及防治效果，并概括了木霉在生防过程中对植物病原物的生防机制，包括竞争、重寄生、抗生作用等，以及木霉与植物互作中对植物促生和诱导植物抗性的机制。目前，世界上含木霉的商品化制剂已超过250种，在不同国家地区都取得了良好的防治效果，更多的优秀生防木霉菌株也在通过野生菌株筛选或遗传改良等方式开发。木霉生物防治及机制研究对推广生物防治和减少化学农药有重要意义。     

The <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> assay: a tool to evaluate the pathogenic potential of bacterial biocontrol agents

Bacterial biocontrol agents (BCAs) open up the possibility of controlling plant pathogens in an environmentally friendly way. Although they are naturally occurring microbes, some of them can cause diseases in humans. For successful registration it is necessary to test potentially adverse effects on the human health of at-risk candidates. Existing pathogenicity assays are cost-intensive, time-consuming and furthermore they are often inappropriate for facultative pathogens. We developed a new, fast and inexpensive bioassay on the basis of the nematode Caenorhabditis elegans, which is a well-accepted model organism to study bacterial pathogenicity. A selection of eight strains from clinical and environmental origin as well as potential and commercial BCAs from the genera Bacillus, Pseudomonas, Serratia and Stenotrophomonas were screened for their potential to kill the nematode in an in vitro agar plate assay. Furthermore, the motility and reproductive behaviour of nematodes exposed to strains were tested in comparison with those fed by the human pathogen Pseudomonas aeruginosa QC14-3-8 (positive control) and the negative control Escherichia coli OP50. Commercial as well as potential biocontrol strains did not display any adverse effects in all tests. In contrast, the C. elegans assay showed slight effects for clinical and environmental Stenotrophomonas strains. Results showed that the nematode C. elegans provides a model system to indicate the pathogenic potential of BCAs in a very early stage of product development.     

Evaluation of biological control agents for managing cucurbit powdery mildew on greenhouse-grown melon

An evaluation was made of the ability of two mycoparasite-based products AQ10® ( Ampelomyces quisqualis ) and Mycotal® ( Lecanicillium lecanii ), as well as three strains of Bacillus subtilis , to manage powdery mildew disease, caused by Podosphaera fusca on melon seedlings maintained under different regimes of relative humidity and on plants grown under greenhouse conditions in Spain. In every case fungal and bacterial biocontrol agents (BCAs) performed better under conditions of high relative humidity (90–95% RH). In greenhouse experiments, the effectiveness of the mycoparasites to manage powdery mildew was absolutely dependent on mineral oil. The strains of B. subtilis provided disease control similar to that achieved with the mycoparasites or the fungicide azoxystrobin. Microscopic analysis showed the ability of these bacterial strains to efficiently colonize leaf surfaces and revealed the occurrence of antagonistic interactions between biological agents and P. fusca structures. These results confirmed the usefulness of these BCAs for managing powdery mildew on greenhouse-grown cucurbits either as single products or as a component of integrated control programmes.     

Interaction of Moniliophthora perniciosa biotypes with Micro‐Tom tomato: a model system to investigate the witches' broom disease of Theobroma cacao

The miniature tomato (Solanum lycopersicum) cultivar Micro‐Tom (MT) has become an important platform to investigate plant–pathogen interactions. In the case of the witches' broom disease of Theobroma cacao (cacao), the existence of Moniliophthora perniciosa isolates pathogenic to Solanaceae (S‐biotype) may enable the use of MT to circumvent limitations of the cacao host, whereas the availability of a non‐infective cacao C‐biotype allows the evaluation of contrasting responses of MT. Infection of MT by the S‐biotype led to stem swelling and axillary shoot growth to form broom‐like symptoms similar to the biotrophic phase in cacao, but the infected tissues did not progress to necrosis. Conversely, inoculation with the C‐biotype did not cause typical symptoms, but reduced plant height, appearing as a non‐host interaction. Histopathological characterization of the S‐biotype infection of MT by light and electron microscopy indicated limited germ tube penetration, preferentially through wounds at the base of trichomes or actively through the epidermis. No intracellular mycelium was observed, corroborating the lack of the necrotrophic stage of the pathogen. The analysis of gene expression during the interaction between the S‐ or C‐biotype with MT indicated that expression of plant defence‐associated genes differs for kinetics and intensity between a compatible or incompatible M. perniciosa–MT interaction. The pattern of spore germination and low rate of mycelia penetration suggests that the S‐biotype is not a fully adapted tomato pathogen, but possibly a case of broken non‐host resistance, and evidence suggests the occurrence of a non‐host MT response against the C‐biotype.     

Work of the EPPO/IOBC Panel on biological control agents

EPPO started work on biological control agents (BCAs) in 1996, and the joint EPPO/IOBC Panel was established in 1997. The history of the Panel is provided and EPPO Standards developed by the Panel are described. These Standards are: PM 6/1 ‘First import of exotic biological control agents for research under contained conditions’, PM 6/2 ‘Import and release of non‐indigenous biological control agents’ and PM 6/3 ‘List of biological control agents widely used in the EPPO region’. The last of these has been annually updated as a ‘Positive List’ of BCAs for which EPPO recommends its member countries to use a simplified procedure for import and releases. EPPO activities in biological control have been focused on the safety aspects of the introduction of invertebrate BCAs. However, the scope of EPPO's work in this area is now under review, and a number of issues are being considered by the Working Party on Phytosanitary Regulations. These include the remit of the Panel and Standards in relation to micro‐organisms, the evaluation of potential environmental benefits as well as potential risks from releases and the potential for use of BCAs against regulated pests and those recommended for regulation.     

PM 6/2 (2): Import and release of non‐indigenous biological control agents

Specific Scope

This standard gives guidelines for the import, release and required application procedures for non‐indigenous biological control agents (BCAs).

Specific approval and amendment

Revision approved in 2010–09.     

Technical and commercial aspects of biocontrol products

The global agrochemical market in 1991 was $26800 million, yet biological products were reported to account for only $120 million of sales per annum—less than 0.5 % of the total. The majority of these sales are attributed to bio-insecticides of which Bacillus thuringiensis accounts for over 90%, but B. thuringiensis could be described as a biologically produced insecticide, rather than a true biocontrol agent. Biological products have technical limitations, including extreme specificity, sensitivity to environmental factors and problems with robustness of the formulations, but ironically, it is these limitations which also give biological control an image of environmental acceptability. Nonetheless, some of the limitations will be overcome and sales will increase, but primarily in niche situations such as the control of soil-borne diseases and the control of insect pests showing resistance to agrochemicals. In order for significant inroads to be made into such niche markets it is imperative that progress with biological products is not impaired by over-regulation, and a rational approach by all regulatory bodies is required. Overall, though, agrochemicals are likely to continue to be the major method of crop protection for the foreseeable future, and the biological control field now needs clear, well-defined goals if current successful niche products can be the basis for future success rather than a limited experiment in alternative technology.     

Biological control of Ralstonia wilt,Phytophthora blight,Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89

In a previous study, we screened the combination of the three bacterial strains—Bacillus subtilis AR12, Bacillus subtilis SM21, and Chryseobacterium sp. R89—(BBC), which can control mixed diseases, including Ralstonia wilt, Phytophthora blight, and Meloidogyne root-knot on bell pepper with high biocontrol efficacy and yield increase under greenhouse and field conditions. In this study, to achieve the best biocontrol agents for separately controlling Ralstonia wilt, Phytophthora blight and Meloidogyne root-knot by BBC, the biocontrol efficacy by BBC-related BCAs (AR12, SM21, R89, AR12 + SM21, AR12 + R89, SM21 + R89 and AR12 + SM21 + R89) were compared regarding all three single diseases and the mixed diseases, respectively. BBC achieved the highest biocontrol efficacy against all three single diseases and the mixed diseases (biocontrol efficacies of 92.46 %, 81.81 %, 86.49 % and 87.31 % against Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot and the mixed diseases, respectively), which were significantly more than that attained by the three single strains and the two-strain combinations AR12 + SM21, AR12 + R89 and SM21 + R89. Although a biomass increase of 4.86–21.14 % was attained by BBC-related BCAs without BBC, BBC achieved a maximum biomass increase of 64.67 %.     

PM 6/2 (3) Import and release of non‐indigenous biological control agents

Specific scope

This Standard gives guidelines for the import, release and required application procedures for non‐indigenous biological control agents (BCAs).

Specific approval and amendment

Revision approved in 2010–09. Second revision approved in 2014–09.     

Combining physical, cultural and biological methods: prospects for integrated non-chemical weed management strategies

Physical, cultural and biological methods for weed control have developed largely independently and are often concerned with weed control in different systems: physical and cultural control in annual crops and biocontrol in extensive grasslands. We discuss the strengths and limitations of four physical and cultural methods for weed control: mechanical, thermal, cutting, and intercropping, and the advantages and disadvantages of combining biological control with them. These physical and cultural control methods may increase soil nitrogen levels and alter microclimate at soil level; this may be of benefit to biocontrol agents, although physical disturbance to the soil and plant damage may be detrimental. Some weeds escape control by these methods; we suggest that these weeds may be controlled by biocontrol agents. It will be easiest to combine biological control with fire and cutting in grasslands; within arable systems it would be most promising to combine biological control (especially using seed predators and foliar pathogens) with cover‐cropping, and mechanical weeding combined with foliar bacterial and possibly foliar fungal pathogens. We stress the need to consider the timing of application of combined control methods in order to cause least damage to the biocontrol agent, along with maximum damage to the weed and to consider the wider implications of these different weed control methods.     

害虫生物防治新技术——载体植物系统

建立一个自我维持并可有效降低害虫种群水平的系统是害虫生物防治长期追求的理想目标。载体植物系统（banker plant system）又称开放式天敌饲养系统,是近年来开发出的一种集保护利用本地天敌、人工繁殖释放天敌以及异地引进天敌等传统技术特点为一体的新型生物防治技术。载体植物（banker plants）、替代食物（alternative foods）和有益生物（beneficial）是该系统的三个基本要素。本文对载体植物和载体植物系统概念、特点以及近年来国际上的研究进展进行了综述,并结合自身的研究实践,举例介绍载体植物系统的应用,以推动国内外对载体植物系统的研究和应用。     

Chemical and biological control of Fusarium species involved in garlic dry rot at early crop stages

European Journal of Plant Pathology - The aim of the study was to test in vitro and in vivo the efficacy of triazoles and biocontrol agents (BCAs) against Fusarium proliferatum and F. oxysporum,...     

Recent studies on biological control of plant diseases in Japan

Microorganisms play an enormously important role in plant disease control. Research on biological control of plant pathogens has received major impetus and attracted many researchers during the past few decades due to the increased public concern on hazards associated with the use of synthetic pesticides. From research on utilizing specific antagonistic microorganisms, many effective biological control agents (BCAs) have been found and are increasingly implemented in integrated pest management strategies to control plant diseases. Here current research results on biological control against plant diseases carried out in Japan are reviewed by focusing on major categories of BCAs: fungi, bacteria and actinomycetes and attenuated viruses.     

No indication of strict host associations in a widespread mycoparasite: grapevine powdery mildew (Erysiphe necator) is attacked by phylogenetically distant Ampelomyces strains in the field

Pycnidial fungi belonging to the genus Ampelomyces are common intracellular mycoparasites of powdery mildews worldwide. Some strains have already been developed as commercial biocontrol agents (BCAs) of Erysiphe necator and other powdery mildew species infecting important crops. One of the basic, and still debated, questions concerning the tritrophic relationships between host plants, powdery mildew fungi, and Ampelomyces mycoparasites is whether Ampelomyces strains isolated from certain species of the Erysiphales are narrowly specialized to their original mycohosts or are generalist mycoparasites of many powdery mildew fungi. This is also important for the use of Ampelomyces strains as BCAs. To understand this relationship, the nuclear ribosomal DNA internal transcribed spacer (ITS) and partial actin gene (act1) sequences of 55 Ampelomyces strains from E. necator were analyzed together with those of 47 strains isolated from other powdery mildew species. These phylogenetic analyses distinguished five major clades and strains from E. necator that were present in all but one clade. This work was supplemented with the selection of nine inter-simple sequence repeat (ISSR) markers for strain-specific identification of Ampelomyces mycoparasites to monitor the environmental fate of strains applied as BCAs. The genetic distances among strains calculated based on ISSR patterns have also highlighted the genetic diversity of Ampelomyces mycoparasites naturally occurring in grapevine powdery mildew. Overall, this work showed that Ampelomyces strains isolated from E. necator are genetically diverse and there is no indication of strict mycohost associations in these strains. However, these results cannot rule out a certain degree of quantitative association between at least some of the Ampelomyces lineages identified in this work and their original mycohosts.     

RNA‐based biocontrol compounds: current status and perspectives to reach the market

Facing current climate challenges and drastically reduced chemical options for plant protection, the exploitation of RNA interference (RNAi) as an agricultural biotechnology tool has unveiled possible new solutions to the global problems of agricultural losses caused by pests and other biotic and abiotic stresses. While the use of RNAi as a tool in agriculture is still limited to a few transgenic crops, and only adopted in restricted parts of the world, scientists and industry are already seeking innovations in leveraging and exploiting the potential of RNAi in the form of RNA‐based biocontrol compounds for external applications. Here, we highlight the expanding research and development pipeline, commercial landscape and regulatory environment surrounding the pursuit of RNA‐based biocontrol compounds with improved environmental profiles. The commitments of well‐established agrochemical companies to invest in research endeavours and the role of start‐up companies are crucial for the successful development of practical applications for these compounds. Additionally, the availability of standardized guidelines to tackle regulatory ambiguities surrounding RNA‐based biocontrol compounds will help to facilitate the entire commercialization process. Finally, communication to create awareness and public acceptance will be key to the deployment of these compounds. © 2019 Society of Chemical Industry