Biological control of bayoud disease in date palm: Selection of microorganisms inhibiting the causal agent and inducing defense reactions

Twenty-one isolates of microorganisms, including Bacillus spp., Rhizobium spp., Ulocladium atrum, Candida guilliermondii, Pseudomonas sp., Rahnella aquatilis and other bacteria not yet identified, were tested to determine their effects on the mycelial growth and the sporulation of Fusarium oxysporum f.sp. albedinis (Foa), the causal agent of bayoud on date palm. The potential of these antagonists in the induction of defense reactions in date palm seedlings was also studied. Four bacteria, B. pumilus W1, R. aquatilis W2, B. cereus X16 and n.d. S1, have exhibited a high inhibition toward mycelial growth of Foa (70–77%), and its sporulation (80–95% of the control). Moreover, cytological alterations have been detected in the Foa mycelium grown in the inhibition zone. Application of these antagonists into date palm seedlings has led to trigger defense reactions with an accumulation of non-constitutive hydroxycinnamic acid derivatives, such as the sinapic derivative I2, known to play a crucial role in resistance of date palm to Foa. This reaction was more pronounced in resistant cultivar (BSTN) than in susceptible (JHL). The combined effects of direct and indirect actions of Foa antagonists are discussed in the hope of providing a biocontrol strategy against bayoud.     

Electrophysiological Responses to Fusaric Acid of Root Hairs from Seedlings of Date Palm-susceptible and -resistant to Fusarium oxysporum f. sp. albedinis

Bayoud, a vascular wilt of date palm caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most devastating disease in palm groves of north Africa. Although Foa is able to induce resistance mechanisms in its host plant, no data are available on the early responses of the root cells. Fusaric acid (FA) is the main toxin found in culture filtrates from Foa aggressive strains. This phytotoxin induces modifications of membrane permeability or membrane potential in various cell types and could thus be involved in the early steps of signal exchange between the pathogen and the plant. We showed an early differential behaviour of the disease‐resistant and ‐susceptible cultivars from date palm when challenged by FA. This response could be due to a differential sensitivity of H⁺‐ATPases to FA.     

Identification of phenylacetic acid produced by Fusarium oxysporum f. sp. albedinis, the causal agent of bayoud, using GC-MS

These studies are concerned with the isolation and identification of secondary metabolites produced by Fusarium oxysporum f. sp. albedinis (F. o. a.), the causal agent of bayoud, the wilt disease of the date palm (Phoenix dactylifera L.). Fungal secondary metabolites are chemical compounds identified in a limited number of species. They consist of toxins, antibiotics and antifungal agents. Among the metabolites we could isolate from the pathogen grown in a liquid medium, and then identify by gas chromatography coupled with mass spectrometry (GC-MS), phenylacetic acid has been distinguished. This compound is widely described in the literature as having antimicrobial, antifungal, phytotoxic properties and also endowed with hormonal activity similar to that of indole acetic acid (IAA). To date, this metabolite has never been reported in F. o. a.     

Relationship between Cell Wall Susceptibility to Cellulases and Pectinases of Fusarium oxysporum and Susceptibility of Date Palm Cultivars

Fusarium oxysporum f. sp. albedinis, the bayoud disease agent of date palm, grows on a mineral medium containing the cell walls of date palm roots as a sole carbohydrate source. The growth and development of pathogen under these conditions was related to the production of extracellular cell wall-degrading enzymes (CWDE): cellulases, polygalacturonases, polygalacturonate transeliminases, and pectinmethylesterases. The mycelial growth and the sporulation of pathogen were higher in the presence of cell walls of susceptible cultivars (BFG, JHL, BSK) than in the presence of those of resistant cultivars (IKL, SLY, BSTN). After 8 d of fungal culture, the activity of CWDE was equal whatever is the origin of the cell walls (resistant or susceptible cultivars). After 16 d of culture, the activity of these CWDE was higher when the parasite was cultivated on the cell walls of the susceptible cultivars than on those of the resistant cultivars. A positive correlation was observed between CWDE activities and the growth and the sporulation of F. oxysporum after 16 d of culture. These results clearly show a relation between the susceptibility/resistance of the cell walls of the roots of the date palm to the parasitic CWDE and the susceptibility/resistance of the cultivars.     

Enhancement of Defence Responses against Bayoud Disease by Treatment of Date Palm Seedlings with an Hypoaggressive Fusarium oxysporum Isolate

Pretreatment of date palm seedlings with an hypoaggressive Fusarium isolate (AHD) protected them partially from further infection by Fusarium oxysporum f.sp. albedinis (Foa), the Bayoud disease pathogen. No mortality occurred during 2–3 months of incubation in plants pretreated with AHD, as opposed to aggressive isolate (ZAG) inoculated controls where up to 100% mortality was observed 15–30 days after inoculation. Such protection involved biochemical interactions between the host plant and AHD since no direct competition or antagonism was revealed between AHD and ZAG. The examination of the accumulation of phenolics and peroxidase activity, two parameters previously reported to be involved in date palm resistance to Foa, indicated that the response to AHD was correlated with the ability of pretreated date palm tissues to establish a faster defence response in the roots of both susceptible and resistant cultivars. Plants pretreated with AHD accumulated higher amounts of phenolics, mainly non‐constitutive hydroxycinnamic acid derivatives, which play a crucial role in date palm defence against Foa, as previously described by our group. These compounds were accumulated along with the constitutive caffeoylshikimic acids. A faster induction of peroxidase activity in response to Foa was also recorded in plants pretreated with AHD. Given the multi‐component nature of these induced responses, AHD could be part of integrated disease management strategies for a sustainable control of the palm tree Bayoud disease.     

Contribution of cell and molecular biology and genetics to plant protection

Plants resist to the majority of their potential aggressors by opposing physical and chemical barriers: cell walls, secondary metabolites.... Phenomena of specific recognition between a plant variety and a pathovar induce on the one hand, a local (hypersensitive) reaction that tends to limit pathogen growth and, on the other hand, a cascade of signals that allows the activation of a non-specific general (systemic) resistance. The contribution of genetics to the fight against pathogens depends on the natural variability that comes from the co-evolution between plants and their aggressors. Many plant varieties resistant to one or several pathogens have been obtained and are cultivated. The use of biotechnology will facilitate the rapid generation of new, resistant cultivars and cultivars with multiple resistances. New methods in order to increase the efficiency and the durability of resistance are envisaged.     

In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens

Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.     

Constraints on evolution of virus avirulence factors predict the durability of corresponding plant resistances

Understanding the factors driving pathogen emergence and re-emergence is a major challenge, particularly in agriculture, where the use of resistant plant cultivars imposes strong selective pressures on plant pathogen populations and leads frequently to 'resistance breakdown'. Presently, durable resistances are only identified after a long period of large-scale cultivation of resistant cultivars. We propose a new predictor of the durability of plant resistance. Because resistance breakdown involves modifications in the avirulence factors of pathogens, we tested for correlations between the evolutionary constraints acting on avirulence factors or their diversity and the durability of the corresponding resistance genes in the case of plant–virus interactions. An analysis performed on 20 virus species–resistance gene combinations revealed that the selective constraints applied on amino acid substitutions in virus avirulence factors correlate with the observed durability of the corresponding resistance genes. On the basis of this result, a model predicting the potential durability of resistance genes as a function of the selective constraints applied on the corresponding avirulence factors is proposed to help breeders to select the most durable resistance genes.     

How to cope with insect resistance to Bt toxins?

Transgenic Bt crops producing insecticidal crystalline proteins from Bacillus thuringiensis, so-called Cry toxins, have proved useful in controlling insect pests. However, the future of Bt crops is threatened by the evolution of insect resistance. Understanding how Bt toxins work and how insects become resistant will provide the basis for taking measures to counter resistance. Here we review possible mechanisms of resistance and different strategies to cope with resistance, such as expression of several toxins with different modes of action in the same plant, modified Cry toxins active against resistant insects, and the potential use of Cyt toxins or a fragment of cadherin receptor. These approaches should provide the means to assure the successful use of Bt crops for an extended period of time.     

Antibody-Based Resistance to Plant Pathogens

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

Interactions of Apple and the Alternaria alternata Apple Pathotype

Apple is one of the most cultivated tree fruits worldwide, and is susceptible to many diseases. Understanding the interactions between the host and pathogen is critical in implementing disease management strategies and developing resistant cultivars. This review provides an update on the interactions of apple with Alternaria alternata apple pathotype, which causes Alternaria blotch, with a brief history about the discovery of the disease and pathogen and its damage and epidemiology. The focus of the review is placed on the physiological and genetic response of the host to pathogen infection, including resistance and susceptibility, and the molecular markers associated with them. Of the response of the pathogen to the host, the emphasis is placed on the role of the selective toxins on pathogenicity and their genetic controls and regulations. The review ends with a perspective on future directions in the research on the apple-A. alternata pathosystem in the era of genomics and post genomics, particularly on how to identify candidate genes from both host and pathogen for potential genetic engineering for disease resistant cultivars.     

A mitochondrial molecular marker of resistance to Bayoud disease in date palm

We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality. Received: 14 August 2000 / Accepted: 10 November 2000     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

Bacterial larvicides for vector control: mode of action of toxins and implications for resistance

Vector control can be an effective strategy to interrupt disease transmission and biolarvicides based on the entomopathogenic bacteria Bacillus sphaericus, and Bacillus thuringiensis serovar israelensis (Bti) have been successfully used to control species of public health relevance from the genera Aedes, Culex, Anopheles and Simulium. The most important feature of these agents is their ability to produce insecticidal proteins with selective action on the larval midgut. These protoxins are produced as crystals that, once ingested by larvae, are processed into active toxins, interact with receptors in the midgut epithelium and trigger cytopathological effects leading to larval death. B. sphaericus and Bti toxins share the initial steps of the mode of action; however, they interact with different midgut molecules. B. sphaericus presents a single larvicidal factor, the binary (Bin) toxin, whose action relies on the binding to one class of midgut receptors, while Bti crystals contain four protoxins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa), which display interactions with multiple midgut receptors. The mode of action of B. sphaericus displays a greater potential for resistance selection, compared to Bti, and, to date, there is no record of insect resistance to the latter, contrarily to B. sphaericus. The set of mosquitocidal toxins and their interaction with midgut target sites are described in this review, as well as the implications for the potential to select resistance amongst exposed populations. These biolarvicides have specific mode of action that rely on unique interactions and make them the most selective agents to control Diptera insects actually available.     

Resistance to Bacillus thuringiensis in the cabbage looper (Trichoplusia ni) increases susceptibility to a nucleopolyhedrovirus

Cabbage loopers, Trichoplusia ni, are pests in many agricultural settings including vegetable greenhouses in British Columbia (Canada), where microbial insecticides based on Bacillus thuringiensis (Bt) toxins are commonly used. Frequent use of these insecticides has led to resistance in some populations. An alternative microbial control is the multiple nucleopolyhedrovirus of the alfalfa looper (Autographa californica), AcMNPV which occurs naturally, but at low frequencies in T. ni populations. Bioassays show that T. ni resistant to Bt were twice as susceptible to AcMNPV as were individuals from the Bt-susceptible strain and AcMNPV could be complementary in a resistance management program for T. ni.     

Conserving the efficacy of insecticides against Plutella xylostella (L.) (Lep., Plutellidae)

Abstract:  The diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), is one of the most destructive insect pests of crucifers worldwide. It was the first crop insect reported to be resistant to DDT and now in many crucifer-producing regions it has shown significant resistance to almost every insecticide applied in field including biopesticides such as crystal toxins from Bacillus thuringiensis and spinosyns from Saccharopolyspora spinosa . In certain parts of the world, economical production of crucifers has become almost impossible because of its resistance to insecticides and resulting control failure. A coordinated resistance management program needs to be implemented with the involvement of pesticide industry, local pesticide regulatory authorities, scientists and farmers. The judicious use of chemicals in conjunction with other control measures (e.g. biological control agents, resistant varieties, proper fertilization rates) is the best way to manage DBM and other pests of cruciferous crops. Introduction of glucosinolate-sulphatase inhibitors as plant-incorporated-products or sprayable material may also lead to a novel pest management strategy.     

Genetics of resistance to the rust fungus Coleosporium ipomoeae in three species of morning glory (Ipomoea)

We examined the genetic basis of resistance to the rust pathogen Coleosporium ipomoea in three host species: Ipomoea purpurea, I. hederacea, and I. coccinea (Convolvulaceae). In crosses between resistant and susceptible individuals, second-generation selfed offspring segregated in ratios that did not differ statistically from the 3:1 ratio indicative of single-gene resistance with the resistant allele dominant. One out of three crosses between resistant individuals from two different populations revealed that resistance loci differed in the two populations, as evidenced by the production of susceptible individuals among the S(2) generation. These results suggest that gene-for-gene interactions contribute substantially to the dynamics of coevolution in this natural pathosystem. They also suggest that evolution of resistance to the same pathogen strain may involve different loci in different Ipomoea populations.     

Animal Models of Seizures and Epilepsy: Past,Present, and Future Role for the Discovery of Antiseizure Drugs

The identification of potential therapeutic agents for the treatment of epilepsy requires the use of seizure models. Except for some early treatments, including bromides and phenobarbital, the antiseizure activity of all clinically used drugs was, for the most part, defined by acute seizure models in rodents using the maximal electroshock and subcutaneous pentylenetetrazole seizure tests and the electrically kindled rat. Unfortunately, the clinical evidence to date would suggest that none of these models, albeit useful, are likely to identify those therapeutics that will effectively manage patients with drug resistant seizures. Over the last 30 years, a number of animal models have been developed that display varying degrees of pharmacoresistance, such as the phenytoin- or lamotrigine-resistant kindled rat, the 6-Hz mouse model of partial seizures, the intrahippocampal kainate model in mice, or rats in which spontaneous recurrent seizures develops after inducing status epilepticus by chemical or electrical stimulation. As such, these models can be used to study mechanisms of drug resistance and may provide a unique opportunity for identifying a truly novel antiseizure drug (ASD), but thus far clinical evidence for this hope is lacking. Although animal models of drug resistant seizures are now included in ASD discovery approaches such as the ETSP (epilepsy therapy screening program), it is important to note that no single model has been validated for use to identify potential compounds for as yet drug resistant seizures, but rather a battery of such models should be employed, thus enhancing the sensitivity to discover novel, highly effective ASDs. The present review describes the previous and current approaches used in the search for new ASDs and offers some insight into future directions incorporating new and emerging animal models of therapy resistance.     

Genes and environment interact to determine the fitness costs of resistance to Bacillus thuringiensis

Genes which provide resistance to novel challenges such as pesticides, toxins or pathogens often impose fitness costs on individuals with a resistant phenotype. Studies of resistance to Bacillus thuringiensis and its insecticidal Cry toxins indicate that fitness costs may be variable and cryptic. Using two field populations (Karak and Serd4) of the diamondback moth, Plutella xylostella, we tested the hypothesis that the costs associated with resistance to the B. thuringiensis toxin Cry1Ac would be evident when insects were grown under poor environmental conditions, namely limited or poor quality resources. On a poor quality resource, a cultivar of Brassica oleracea var. capitata with varietal resistance to P. xylostella, only one resistant population, Karak, showed reduced fitness. Conversely, when we limited a high quality resource, Brassica pekinensis, by imposing larval competition, only resistant Serd4 insects had reduced survival at high larval densities. Furthermore, Cry1Ac resistance in Serd4 insects declined when reared at high larval densities while resistance at low densities fluctuated but did not decline significantly. These results confirm the hypothesis that resistance costs can appear under stressful conditions and demonstrate that the fitness cost of resistance to Bacillus thuringiensis can depend on the particular interaction between genes and the environment.