Cherry and plum are prone to brown rot diseases. Synthetic fungicides are still the main management tool to control brown rot but there is a need to develop eco-friendly methods. Five commercial biopesticides including BioPK (Trichoderma viride, T. harzianum and T. longibranchum), BoniProtect (Aureobasidium pullulans), Trianum (T. harzianum), PreStop (Gliocladium catenulatum) and Serenade (Bacillus subtilis) along with Indar (fenbuconazole, a synthetic fungicide) were tested against Monilinia laxa which products applied before and after pathogen inoculation. Indar completely inhibited brown rot development. Serenade partially inhibited M. laxa when applied before pathogen inoculation. The efficacy of two indigenous strains (B. subtilis B91 and A. pullulans Y126) were evaluated under ambient conditions or 1°C; Indar and Serenade were used as control treatments. Cherry and plum were first treated with specific products, then wounded and inoculated with M. laxa. Indar completely inhibited M. laxa under both conditions. Under ambient conditions, only B91 reduced the incidence of disease. When stored at 1°C for 4 weeks, all biocontrol treatments reduced the incidence of disease. However, when fruits were left under ambient conditions for an additional 1 week, the incidence of disease did not differ between treatments. Post-harvest application of biopesticides under 1°C was studied with Rovral used as a control. Cherry and plum were submerged into individual treatments and stored at 1°C for 6 weeks. Rovral reduced the incidence of disease, whilst all biopesticides did not. In conclusion, Serenade, B91 and Y126 can partially inhibit M. laxa when applied before pathogen infection. The present study supports that these biocontrol proeducts should be used pre-harvest to reduce the extent of fruit latent infection by M. laxa, and an effective post-harvest cold chain management should be implemented to delay/minimise rot development.

The mycorrhiza and, more generally, soil microbiology research communities recently have lost one of their most ardent scientists. José Miguel Barea was a world leader of arbuscular mycorrhiza research and pioneered the establishment of such studies in Spain and Latin American. He was a prolific publisher, enthusiastic teacher of many graduate students and a genial host to visitors of his beloved Granada. He will be missed wherever mycorrhizasts gather.

Strawberry is an important fruit crop within the UK. To reduce the impact of soil-borne diseases and extend the production season, more than half of the UK strawberry production is now in substrate (predominantly coir) under protection. Substrates such as coir are usually depleted of microbes including arbuscular mycorrhizal fungi (AMF) and consequently the introduction of beneficial microbes is likely to benefit commercial cropping systems. Inoculating strawberry plants in substrate other than coir has been shown to increase plants tolerance to soil-borne pathogens and water stress. We carried out studies to investigate whether AMF could improve strawberry production in coir under low nitrogen input and regulated deficit irrigation. Application of AMF led to an appreciable increase in the size and number of class I fruit, especially under either deficient irrigation or low nitrogen input condition. However, root length colonization by AMF was reduced in strawberry grown in coir compared to soil and Terragreen. Furthermore, the appearance of AMF colonizing strawberry and maize roots grown in coir showed some physical differences from the structure in colonized roots in soil and Terragreen: the colonization structure appeared to be more compact and smaller in coir.

The effect of inoculation with two arbuscular mycorrhizal fungi (AMF) on growth and drought tolerance of cultivated strawberry ( Fragaria × ananassa ) was studied. Three treatments (a single treatment either of Funneliformis mosseae BEG25, Funneliformis geosporus BEG11 or a 50:50 mixed inoculation treatment of both species) were compared to uninoculated plants. Species-specific primers for qPCR quantification of F. geosporus and F. mosseae DNA were developed to quantify the relative abundance of each fungus in roots of strawberry under different conditions of water stress. Co-occupation of the same root by both species was shown to commonly occur, but their relative abundance varied with water stress (reduced irrigation of up to 40 %). Greater root colonisation was observed microscopically under water stress, but this increased colonisation was often accompanied with decreased amounts of fungal DNA in the root. F. mosseae tended to become more abundant under water stress relative to F. geosporus . There was significant correlation in the fungal colonisation measurements from the microscopic and qPCR methods under some conditions, but the nature of this relationship varied greatly with AMF inoculum and abiotic conditions. Single-species inoculation treatments gave similar benefits to the host to the mixed inoculation treatment regardless of irrigation regime; here, amount of colonisation was of greater importance than functional diversity. The addition of AMF inocula to plants subjected to reduced irrigation restored plant growth to the same or higher values as the non-mycorrhizal, fully-watered plants. The water use efficiency of plants was greater under the regulated deficit irrigation (RDI) regime and in AMF-inoculated plants, but there were no significant differences between plants inoculated with the single or combined inoculum. This study demonstrated that the increase in plant growth was directly influenced by an increase in root colonisation by AMF when individual plants were examined.

Stone fruit is attacked by various pathogens, of which brown rot disease is one of the important diseases. There are three Monilinia species mainly responsible for the brown rot disease: Monilinia fructicola is mainly found in North America and Australasia, and M. laxa and M. fructigena mainly in Europe. Both M. fructicola and M. laxa can infect flowers, resulting in blossom blight, as well as both healthy and wounded fruit, resulting in brown rot. On the other hand, M. fructigena can only infect wounded fruit. Compared to the two other species, M. fructicola has been extensively studied, whereas the equally important M. laxa has had less attention. This paper addresses this imbalance and reviews research on the biology, epidemiology and management of M. laxa on stone fruits. Due to EU regulations, the number of fungicides available for controlling plant diseases has been steadily decreasing, particularly in the post-harvest environment. This has placed much more emphasis on alternative control methods, a focus of the present review. Numerous physical and biological approaches to control have achieved successful outcomes but often in small-scale trials and in isolation from integrated strategies. Promising physical control methods include removal of mummified fruit in orchards and post-harvest hot-water treatment. Many micro-organisms have been shown to have biocontrol potential against brown rot but only a few have been commercially formulated. It is generally agreed that the use of biocontrol agents needs to be integrated with other measures. Current research focuses on disease management from flowering to post-harvest period. Recent results have suggested that reducing overwintering inoculum should be considered as one of key aspects of integrated management of brown rot on stone fruit. Finally, we make recommendations about future research and development on integrated pest management strategies for control of M. laxa , especially on strategic deployment of biocontrol agents and interactions among brown rot pathogens.

Brown rot on cherry and plum, caused by Monilinia laxa, is an important disease, for which overwintered mummified fruit is a significant inoculum source for infection of flowers and fruit in the spring. Experiments were conducted to assess the potential of applying plant protection products in winter and/or early spring to suppress sporulation on mummified fruit. Products tested included Indar 5 EW (a commercial fungicide, a.i. fenbuconazole), Aureobasidium pullulans Y126 [a candidate biocontrol agent (BCA)]), Bacillus sp. B91 (a candidate BCA) and Serenade (a commercially formulated BCA of Bacillus subtilis strain QST 713) in addition to control treated with tap water. Indar and A. pullulans Y126 significantly reduced sporulation when applied once in winter. Overall, a single treatment in early spring (February) was slightly more effective than single treatment in winter (November). Application of all products in both winter and early spring led to significant reduction in sporulation. Indar had the highest efficacy, reducing the number of spores from 9 × 105 to 5 × 103 per mummified plum when applied twice. Of the three BCAs applied on both occasions, A. pullulans Y126 had the highest efficacy, reducing the number of spores from 9 × 105 to 5 × 104 per mummified plum. There were no synergistic but additive effects between the two applications in winter and early spring based on the Bliss independence test. These results suggest that reducing overwintering inoculum in dormant season is effective and may be part of an integrated management strategy for brown rot on stone fruit.

Plant-microbe symbioses are common, ubiquitous, and very varied. The health and vigor of plants is dependent on the multifarious relationships they have with symbiotic microbes. Many of these symbioses are ancient and essential for plants to survive in natural ecosystems, where critical nutrients are often limiting or pathogen populations are highly active. Coevolution of plants and their associated microbial symbioses have been a key driver of the evolutionary process. Recently it has become acknowledged that many plants form such relationships and harbor beneficial microbes within both their root systems and their aerial tissues. As with mycorrhizal fungi, the coevolution of many plants and their associated endosymbionts has resulted in an interdependence that now means that neither partner can flourish alone. This chapter discusses diversity within prokaryotic and eukaryotic symbioses. Many soil fungi form joint organs with plants called mycorrhizas. The easiest to observe are ectomycorrhizas formed on the short roots of most temperate forest trees. A high proportion of these undescribed species will form loose or strong symbiotic relationships with plants. The shift in phylogenetic approaches emphasized in this book has opened a myriad of opportunities for new discoveries. Thus conservation strategies are significant for their retention of plant-microbial symbioses as well as for their intrinsic protection of the plants themselves.

Monilinia laxa is the causal agent of brown rot disease on stone fruits, and also causes blossom wilt and twig canker. The common practice used to manage this disease is through fungicide treatments. However the demand to reduce fungicide inputs has been increasing and there is a growing number of reports of M. laxa strains that are resistant to fungicides. There is an urgent need to search for an alternative strategy to control the disease. This study focused on the isolation and characterisation of biological control agents (BCAs) using indigenous isolates isolated from cherries and plums collected within the UK. A total of 192 isolates were screened against two strains of M. laxa in a series of in vitro dual culture tests. From this in vitro screen, 12 isolates were selected for a subsequent in vivo screen on detached fruits, which then narrowed these isolates down to two potential BCAs. These two strains were identified as Bacillus amyloliquefaciens/subtilis (isolate B91) and Aureobasidium pullulans (isolate Y126). The capability of these two potential BCAs to grow and survive at a range of temperatures likely to be experienced under field and storage conditions was studied in order to gain knowledge for product formulation and field application. Bacillus sp. B91 was shown to be a mesophilic bacterium that could grow at 10–25 °C but suffered significant mortality at 0 and 5 °C, while A. pullulans Y126 was both mesophilic and psychrotolerant as it grew between 0–25 °C with the optimum at 20 °C. When all nutrients were removed, Y126 was able to survive for several weeks in all test temperatures (0–25 °C) but showed significant mortality at 25 °C. The capability of B91 to survive at 20 and 25 °C was higher than at low temperatures (0–15 °C). In addition, the modes of action of the potential BCAs were studied. B91 was shown to produce soluble and volatile organic compounds that inhibited M. laxa, while A. pullulans Y126 did not produce inhibitory compounds, but appeared to inhibit the pathogen via competition for nutrients. This study shows that microbial antagonists against M. laxa can be found from indigenous sources and that they are capable of preventing brown rot disease in controlled conditions, thus demonstrating a potential to be developed into commercial products.

The diversity of Fusarium populations in asparagus (Asparagus officinalis L.) decline fields in Japan was estimated by PCR-SSCP (single-stranded conformational polymorphism) analysis of the ITS2 regions of the nuclear rRNA genes. This method was used to rapidly and objectively identify pathogens associated with roots of plants showing symptoms of asparagus decline collected from fields in five regions across Japan. Over 651 fusarial isolates were obtained, and were easily differentiated into three principal species. Fusarium oxysporum f. sp. asparagi was most frequently isolated from the domestic five regions (68%), whereas Fusarium proliferatum (28.6%) was less frequent. Fusarium solani was found much rarely (2.5%). The frequency of isolation of Fusarium proliferatum increased gradually from the north to the south of Japan, though considerable differences were found between fields in each region, as well as regional differences among the Fusarium populations. Most of the fusarial isolates were highly pathogenic in vitro. These results reveal that Fusarium oxysporum f. sp. asparagi and Fusarium proliferatum are important biotic factors which lead to asparagus decline in Japan.

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.

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.

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.

Abstract Experiments were conducted with Botrytis cinerea on strawberry leaves to investigate where combinations of commercially available biological control agents (BCAs) might control B. cinerea more effectively than individual BCAs. Specifically, we studied the persistence of biocontrol activities, spread of BCAs among leaves, and biocontrol efficacy in relation to application regimes: mixed versus single BCA, pre-versus post-inoculation application, and sequential versus simultaneous application. Three BCA products (Sentinel, Serenade and Trianum) were used for this study. Overall, Serenade did not significantly reduce sporulation of B. cinerea on strawberry leaf discs whereas Sentinel and Trianum gave a similar and significant biocontrol efficacy. Biocontrol efficacy remained almost unchanged 10 days after application at 20/20°C (day/night) or 24/16°C temperature regimes. In contrast, reduced biocontrol efficacy at 26/14°C suggests BCA survival was reduced under these conditions. Incidence of B. cinerea sporulation on leaf discs was ca. 60% higher on leaves that emerged after the BCA application than on leaves directly exposed to BCA, indicating insufficient amount of the BCA had managed to spread to new leaves. Combinations of BCAs, whether applied simultaneously or sequentially (48 h apart), did not improve disease control over the most effective BCA within the combination applied alone. This indicated possible antagonism or interference between the BCAs. Results suggested that there was significant antagonism for most combinations of the three BCAs tested and the degree of antagonism increased as the time from BCA application to pathogen introduction lengthened.

Genetic, phenotypic and functional variation in a Glomus geosporum isolate obtained from a highly alkaline anthropogenic sediment was investigated. Two cultivation lineages of G. geosporum (BEG199 and BEG211) were created by sub-culturing with or without the stress of the sediment. Genetic variation was assessed on spores from both cultivation lineages in the large sub-unit rRNA gene D2 region using PCR-single strand conformational polymorphism (PCR-SSCP) and sequencing. Phenotypical and functional variation of the cultivation lineages were assessed after inoculation onto Conyza bilbaoana. The sequence diversity obtained in G. geosporum BEG211 was significantly different from that obtained in G. geosporum BEG199. Glomus geosporum BEG199 was more effective than G. geosporum BEG211 in promoting the growth of C. bilbaoana in inert substrate and in increasing its leaf phosphorus concentration when the plant was grown in sediment. After inoculation onto C. bilbaoana, G. geosporum BEG199 colonised the roots to a greater extent, produced a significantly larger number of spores and presented a greater length of extraradical mycelium than G. geosporum BEG211. The results indicate that environmental conditions under which arbuscular mycorrhizal fungi (AMF) are cultivated can influence their effects in host plants. Also, AMF might quickly lose gained-tolerance to environmental stresses when maintained without the selective pressure of those stresses. This study has implications for the production of commercial inoculum of AMF and for the maintenance of AMF cultures. The results indicate that inoculum of AMF should be produced and AMF cultures should be maintained in substrates or media containing the original edaphic stresses.

The Petri-dish microcosm is a simple system to study the development of arbuscular mycorrhizal fungi (AMF) and has been shown to be suitable to observe the establishment of Acaulospora tuberculata BEG41, Gigaspora rosea BEG111, Glomus manihotis BEG112 and Scutellospora heterogama BEG40 on plant root. The non-agar based system could be easily used to observe the whole course of development of fungal-root interactions as well as the extra-radical structures of AMF without destructive sampling. It was found that these AMF species were unique with regard to spore germination, root colonization, the architecture of the extra-radical mycelium (ERM), the spread of the ERM in the media, and sporulation. The ERM of A. tuberculata BEG41and G. manihotis BEG112 spread mainly close to the root system, while the ERM of G. rosea BEG111 and S. heterogama BEG40 could occupy areas unoccupied by roots. The unique structure, the branch absorbing structure, was found in the four species of AMF, but differed in their shape and size

A previously published generic mathematic model has been used in a numerical study to understand the dynamics of foliar pathogens in relation to mechanisms, and timing and coverage of biocontrol agent (BCA) applications. With the model parameter values used, it was demonstrated that a BCA possessing either competition or induced resistance as the main mechanism of biological control was more effective in reducing disease development than a BCA with either mycoparasitism or antibiosis as its mechanism. Application coverage, ranging from 50 to 90%, had little effect on biocontrol efficacy, particularly for a BCA with competition and induced resistance as the main mechanism of biocontrol. Conversely, delayed application of BCA had more profound effects on biocontrol efficacy for those with competition or induced resistance as their main mechanism than those with mycoparasitism and antibiosis. Biocontrol efficacy was greatest for a single BCA combining competition with mycoparasitism or antibiosis. The efficacy for a single BCA combining induced resistance with competition critically depended on application time; the efficacy was greatly reduced for delayed applications. The present study suggests that development of an effective strategy for BCA application is critically dependent upon our quantitative understanding of several key biocontrol processes and their interactions. Without reliable quantitative estimation of these processes, it is impossible to make quantitative predictions about biological control and hence to optimize BCA application strategies.

Population studies of arbuscular mycorrhizal fungi (AMF) have traditionally been achieved by indirect analyses of soil-borne spore populations. These studies are not necessarily reflective of populations of AMF within the roots. Advances in molecular biology have revolutionized the analysis of fungal populations colonizing roots and forming mycorrhizas. Initially these studies were qualitative and reported presence or absence of particular AMF species in soils or in roots for comparison between different environments. More recently, the methodology has developed for direct quantification of AMF within roots. Quantitative PCR provides the means to study spatial distribution and individual quantification of AMF in mixed communities over time. In this review, we discuss the progress and application of indirect, direct and finally quantitative methodologies for studying arbuscular mycorrhizal communities. We conclude that the molecular tools now exist to quantitatively analyse the effect of environment, management or inoculation of soils on AMF communities within roots.

Oil palm is a significant and developing crop in many developing countries. The introduction of oil palm puts pressure on natural resources because it is often planted in cleared-cut land that previously supported other crops or was forested. This has led to environmental concerns which require attention. Hence it is important that new plantations are managed in a sustainable way to reduce the impact of oil palm cultivation on ecosystems whilst maximising yield and productivity to farmers. The application of arbuscular mycorrhizal fungi (AMF) technology is one option that can benefit both agronomic plant health and ecosystems. AMF have the potential to increase conventional agricultural productivity and are crucial for the sustainable functioning of agricultural ecosystems. This paper provides an insight into how AMF application might benefit oil palm cultivation through more sustainable management and the practical use of AMF for oil palm plantations.

Experiments were conducted using combinations of commercially available biological control agents (BCAs) to control infection of detached strawberry leaves by Botrytis cinerea Pers.;Fr. Combinations were tested either as mixtures, or as sequential treatments before and after disease loading. Five BCAs were initially tested, giving a range of mechanisms of antagonism. Only three (Sentinel, Serenade and Trianum) were effective in single agent experiments and were taken forward for combinatorial tests. Biocontrol efficacies, relative to the control, varied considerably among replicate trials, but all treatments involving Sentinel (BCA: Trichoderma atroviride P. Karsten LC52) and Trianum (BCA: Trichoderma harzianum Rifai T22) were very effective, either in sequential combination with other BCA or alone. Serenade (BCA: Bacillus subtilis (Ehrenberg) Cohn) was least effective, and sometimes did not result in any significant reduction in disease. Combinations of BCAs as mixtures resulted in less control (i.e., higher disease) than when the most effective BCA within the combination was applied alone, indicating possible antagonism between the BCAs. However, when two BCAs were applied sequentially about 48 h apart, there was no strong evidence for antagonism between the BCAs; this was also confirmed in an experiment using whole plants in controlled environment cabinets. We conclude that there was usually no benefit in applying two BCAs either together or sequentially, so in general we would not advise combinations as the method of choice. Thus, research is necessary to investigate possible interference among specific BCAs, since several BCAs may be applied simultaneously under field conditions to control several diseases.