Biocontrol of phytobacteria with bacteriophage cocktails

Crop loss due to plant pathogens has provoked renewed interest in bacteriophages as a feasible biocontrol strategy of plant diseases. Phage cocktails in particular present a viable option for broadening the phage host range, limiting the emergence of bacterial resistance while maintaining the lytic activity of the phages. It is therefore important that the design used to formulate a phage cocktail should result in the most effective cocktail against the pathogen. It is also critical that certain factors are considered during the formulation and application of a phage cocktail: their stability, the production time and cost of complex cocktails, the potential impact on untargeted bacteria, the timing of phage application, and the persistence in the plant environment. Continuous monitoring is required to ensure that the efficacy of a cocktail is sustained due to the dynamic nature of phages. Although phage cocktails are considered as a plausible biocontrol strategy of phytobacteria, more research needs to be done to understand the complex interaction between phages and bacteria in the plant environment, and to overcome the technical obstacles. © 2019 Society of Chemical Industry     

Association of ‘Candidatus Liberibacter asiaticus’ root infection,but not phloem plugging with root loss on huanglongbing‐affected trees prior to appearance of foliar symptoms

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem‐limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem‐limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem‐plugging as previously hypothesized. The role of root infection in systemic insect‐vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem‐limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem‐limited bacteria to gain greater understanding of how these pathogens cause disease and spread.     

Surfactants in plant disease management: A brief review and case studies

In plant disease management, surfactants are generally used in combination with pesticides to facilitate delivery of the pesticide chemicals to the target sites in plants, vectors, or pathogens. Their intended use is rarely for direct effect on plant pathogens. However, trends in recent studies have revealed an appreciable increase in their use for direct management of plant diseases. In this paper, we briefly review the progress made in the use of surfactants for disease management, as adjuvants and when applied for direct effect. We carried out experiments to evaluate the effect of selected synthetic surfactants (Cohere, Kinetic, Silwet-77, and Induce) on powdery mildew in squash and bacterial spot in tomato. Results from this study showed that the effect of surfactants on these pathogens is dependent on the type of plant pathogen or disease. All tested surfactants exhibited in vitro bactericidal activity against Xanthomonas perforans, a causal pathogen of bacterial spot of tomato. In the greenhouse, Cohere significantly reduced disease severity of powdery mildew in squash compared to the untreated control. Cohere also showed promise in field trials against bacterial spot of tomato, but the other surfactants tested promoted this disease. Our studies demonstrate the importance of evaluating surfactants for their use in management of plant diseases both for direct effect and as adjuvants in tank mix.     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.     

Studies on the biological control of plant pathogens in Finland1

Studies on biological control of plant pathogens in Finland were started by Pohjakallio in the 1950s, concentrating especially on the microbes parasitizing fungal sclerotia. At the turn of the 70s, Kallio studied the protective effect of Finnish isolates of Phlebia gigantea against the most important forest pathogen Heterobasidion annosum with positive results. The preparation was put on the market. Later in the 1970s, the observation by Tahvonen that Finnish light-coloured peat had an inhibitory effect on the infection of plants by some seed and soil-borne fungal pathogens led to further investigations. Of the microbes isolated from peat, Streptomyces spp. proved the most effective antagonists in both in vitro and in vivo tests. Studies are in progress with S. griseoviridis and several other fungal and bacterial antagonists as potential biocontrol agents against some seed and soil-borne pathogens and storage diseases.     

Coffee bacterial diseases: a plethora of scientific opportunities

Coffee is a very important crop for several tropical countries across different continents. The diseases bacterial halo blight (BHB), bacterial leaf spot (BLS), bacterial leaf blight (BLB) and coffee leaf scorch (CLS), caused by the bacterial pathogens Pseudomonas syringae pv. garcae (Psgc), P. syringae pv. tabaci (Psta), Pseudomonas cichorii (Pch) and Xylella fastidiosa subsp. pauca (Xfp), respectively, cause significant reductions in coffee production, although other minor bacterial diseases have also been reported in some countries. Little research progress has been made on aspects that are relevant for control and management of these diseases. In all cases, there is an urgent need to develop rapid and more reliable methods for early detection of the pathogens in order to minimize their negative impact on coffee production. Because of the high rate of intra- and intersubspecific recombination occurring in X. fastidiosa, a permanent revision of the detection methods is necessary. Greater efforts should be made to understand the genetic and virulence diversity of Psgc, Psta and Pch populations. Early studies reported the identification of potential sources of resistance against Psgc and Psta, but, to date, no resistance gene has been isolated. Little effort has been made to understand the biology and molecular mechanisms underlying the interaction between Coffea spp. and these pathogenic bacteria. This review discusses the recent progress on the molecular mechanisms used by these bacteria to cause diseases on other plant species, in order to provide a guideline for the establishment of future research programmes.     

Simultaneous Infection by Pseudomonas syhngae van Hall and Cytospora cincta Sacc. on Apricots

Simultaneous inoculation with Pseudomonas syringae van Hall and Cytospora cincta Sacc. induced more extensive cankers than those caused by either pathogen alone. Freshly cut wounds remained susceptible to cytosporal infection for a longer period than to bacterial inoculation. Knowledge gained of the annual cycle of cytosporal and bacterial diseases offers possibilities for effective control against both pathogens.     

Why many phytophthora root rots and crown rots of tree and horticultural crops remain undetected1

Of the approximately 50 recognized Phytophthora spp., more than half have not been reported to cause root rot or crown rot. Some well-known phytophthora root and crown rots have still not been diagnosed in many regions or countries in the world. It seems likely that this arises mainly from use of inappropriate isolation media and diagnostic procedures. Thus, various root disorders of tree and horticultural crops have been attributed to other pathogens or to abiotic causes, and found to be due to Phytophthora spp. only when proper isolation techniques involving baiting and selective media were used. These special techniques, however, are still not widely used by many diagnosticians and pathologists today. It seems that most pathologists lack training and specific knowledge on Phytophtohora diseases and tools for their study. They are often unaware of the importance of these fungi in causing below-ground infections, or of the unique nature of their biology, soil ecology and pathology in relation to root and crown rot development. It is hoped that wider use of special isolation techniques in routine diagnoses in the future will result in an increase in the frequency of reports of occurrence of phytophthora root and crown rots of woody perennials around the world.     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.     

Ralstonia solanacearum causing potato bacterial wilt: host range and cultivars’ susceptibility in Rwanda

Members of the Ralstonia solanacearum species complex (RSSC), causing potato bacterial wilt or brown rot, are highly contagious and there are no known cultivars with durable resistance to the pathogen. This study hypothesized (a) that crops intercropped or rotated with potato, plants in the same family, and plants grown in the neighbouring fields can host the pathogen and they can be potential sources of primary inoculum, and (b) that potato cultivars currently multiplied by the public tissue culture laboratory in Rwanda are less susceptible to the pathogen. Fourteen plant species and potato, and nine potato cultivars were tested for susceptibility to an RSSC phylotype II strain under greenhouse conditions. The bacteria induced symptoms on potato, tomato, tree tomato, sweet pepper, and eggplant only. Among the plant species with symptoms, potato, tomato, and tree tomato wilted completely. There was a significant difference in days to symptom expression and to complete wilting (p < .0001). While all tested potato cultivars were found to be susceptible, the number of days to first symptom expression, days to complete wilting, area under the disease progress curve (AUDPC), and the number and weight of harvested tubers varied considerably. Cultivars Cruza, Kinigi, and CIP-58 were less susceptible whereas the cultivars Gikungu, Kirundo, and Victoria were highly susceptible. There is a strong need to search for other sources of resistance. The results indicate that some plant species that might serve as a reservoir of the bacterium should be avoided in the vicinity of potato crops.     

Possibilities of avoidance and control of bacterial plant diseases when using pathogen-tested (certified) or -treated planting material

Testing of planting material for freedom from phytopathogenic bacteria is an important, although not exclusive, method for control of bacterial diseases of plants. Ideally, pathogen-free or pathogen-/disease-resistant planting material is desirable, but this situation is not always possible on a practical basis. For most bacterial pathogens, resistance is not available in cultivated hosts, and production of pathogen-free planting material requires strict certification schemes via several routes. These include (i) indexing, with subsequent removal of infected/contaminated material from the production chain; (ii) meristem and other tissue culture production systems; (iii) thermo- or chemotherapy; (iv) plant or seed surface disinfection for epiphytic bacterial pathogens; (v) avoidance or decontamination of contaminated production factors such as substrate, soil or irrigation water. These methods cannot guarantee 100% freedom from the pathogen or disease during crop multiplication from certified planting material, because of factors such as sampling error, experimental error, test sensitivities, limitations of therapies (e.g. phytotoxicity or insufficient penetration), re-introduction of the pathogen, insufficient hygiene or decontamination during planting and multiplication of clean propagating material, and manipulations during trade and production. These factors are discussed with reference to several bacterial plant diseases, in particular control of bacterial brown rot and ring rot of potato in Europe and North America. The most efficient control of bacterial diseases can be expected through a combination of the use of healthy/tested planting material and good cultivation practice, including strict crop and storage hygiene.     

A paucity of bacterial root diseases: Streptomyces succeeds where others fail

There are relatively few bacterial diseases of roots, in comparison to those of aerial plant tissues. Numerous species and pathovars of Pseudomonas,Erwinia and Xanthomonas are important pathogens of leaf and stem tissue on dozens of plant families but these bacterial genera only infrequently attack roots or other underground plant structures. In contrast, there is a growing list of Streptomyces species that are very effective root pathogens. These filamentous, Gram-positive bacteria can cause scab, rot and gall diseases of plant roots and other underground plant structures. The best known pathogenic Streptomyces species is S. scabiei. Horizontal transfer of pathogenicity genes among diverse scab-causing streptomycetes appears to explain the emergence of several new plant pathogens over the last half century. It is proposed that the ability to penetrate plant tissue is essential for successful root infection as there are few natural openings in roots. In contrast, leaves have many natural openings that allow bacteria access to the interior tissues. Thaxtomin, a phytotoxin produced by many plant pathogenic streptomycetes, appears to aid penetration of developing plant tissues by inhibiting primary cell wall development.     

Direct and host‐mediated interactions between Fusarium pathogens and herbivorous arthropods in cereals

Fusarium head blight and fusarium ear rot diseases of cereal crops are significant global problems, causing yield and grain quality losses and accumulation of harmful mycotoxins. Safety limits have been set by the European Commission for several Fusarium‐produced mycotoxins; mitigating the risk of breaching these limits is of great importance to crop producers as part of an integrated approach to disease management. This review examines current knowledge regarding the role of arthropods in disease epidemiology. In the field, diseased host plants are likely to interact with arthropods that may substantially impact the disease by influencing spread or condition of the shared host. For example, disease progress by Fusarium graminearum can be doubled if wheat plants are aphid‐infested. Arthropods have been implicated in disease epidemiology in several cases and the evidence ranges from observed correlations between arthropod infestation and increased disease severity and mycotoxin accumulation, to experimental evidence for arthropod infestation causing heightened pathogen prevalence in hosts. Fusarium pathogens differ in spore production and impact on host volatile chemistry, which influences their suitability for arthropod dispersal. Herbivores may allow secondary fungal infection after wounding a plant or they may alter host susceptibility by inducing changes in plant defence pathways. Post‐harvest, during storage, arthropods may also interact with Fusarium pathogens, with instances of fungivory and altered behaviour by arthropods towards volatile chemicals from infected grain. Host‐mediated indirect pathogen–arthropod interactions are discussed alongside a comprehensive review of evidence for direct interactions where arthropods act as vectors for inoculum.     

Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples

Potato can be infected with many bacterial pathogens, the detection of which is necessary in seed certification. In this study, a diagnostic microarray previously tested for specificity of probes for detecting the potato bacteria causing blackleg and soft rot (Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya spp.), ring rot (Clavibacter. michiganensis subsp. sepedonicus), scab (Streptomyces scabies and Streptomyces turgidiscabies) and brown rot (Ralstonia solanacearum) from pure culture was evaluated for analytical sensitivity when testing directly from tuber samples. The microarray readily detected all the bacterial species when 100 ng of the target bacterial DNA from pure culture was mixed with DNA from soil microbes and potato. However, detection was inconsistent when total DNA isolated directly from infected tubers or enriched bacterial culture was used. While the high specificity of the probes could be confirmed from the results of the DNA cocktail experiment used as a control, the study demonstrated that the level of analytical sensitivity of the microarray under the tested condition was not sufficient to detect bacteria directly from tubers. Therefore, in addition to the cost and organizational complexities, the low analytical sensitivity and limited reproducibility of the microarray are constraints for establishing the platform for routine detection of potato bacterial pathogens from tuber samples.     

Limited asymptomatic colonization of apple tree shoots by Neonectria ditissima following infection of leaf scars and pruning wounds

European apple canker, caused by Neonectria ditissima, is an important disease of apple (Malus domestica). The fungus may reside in the tree without causing symptoms for up to a few years, thus making canker control difficult. Asymptomatic infections established in the nursery can result in severe canker outbreaks in newly established apple orchards. It has been suggested that N. ditissima might colonize the tree beyond the infection point during the asymptomatic stage. We investigated whether N. ditissima can colonize the internal tissues of apple shoots, both prior to and after visual symptoms. Apple trees were artificially inoculated via pruning wounds and leaf scars; then the pathogen was tracked at the inoculation point and beyond with isolation or real-time quantitative PCR (qPCR). Before visual symptoms, N. ditissima could be detected in the infected pruning cut or leaf scar, but not at a distance of 10–15 mm from the entry point, or greater. Conversely, after symptom expression, the pathogen could be detected in the symptomless tissue at 10–15 mm from a canker lesion. This study demonstrated that the asymptomatic infection by N. ditissima can be detected using qPCR and that the pathogen does not grow systemically much beyond the initial entry point inside the plant before visual canker symptoms appear.     

Characterization of Pectolytic Erwinias as Highly Sophisticated Pathogens of plants

Erwinia carotovora and Erwinia chrysanthemi are the two most important soft rotting bacteria of commercially-grown plants. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribsomal gene cluster. Subpopulations grouped into biovars, pathovars, or subspecies associated with various hosts and in different geographic regions suggest specialization in host preference and/or survival in diverse environments. Previous characterization of the pectolytic erwinias as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. The presence of an entire hrp gene cluster and associated type III secretion system, and global regulators which regulate virulence determinants such as exoenzyme production and motility, attest to a highly specialized pathogen. The fact that production of extracellular plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum-sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness.     

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.     

Pathogenicity of fungi and bacteria from the Sudan to water hyacinth

Studies were made in the Sudan for microorganisms pathogenic to water hyacinth (Etchhornia crassipes Solms) for biological control purposes. Many fungi and bacteria were isolated from plant parts showing disease symptoms. Pathogenicity tests revealed that only five out of the twenty-five fungal and bacterial isolates were damaging to the weed plant. These included Acremonium zonatum, Drechslera specifera and Fusarium equiseti. Phoma sorghina and a bacterium Bacillus sp. are reported for the first time as potential pathogens of water hyacinth. Pathogenic isolates were also found to produce toxic metabolites in liquid cultures.