Role of stressed mango host conditions in attraction of and colonization by the mango bark beetle Hypocryphalus mangiferae Stebbing (Coleoptera: Curculionidae: Scolytinae) and in the symptom development of quick decline of mango trees in Pakistan

The mango sudden death syndrome has become a serious threat to the mango industry and caused significant decline in mango production worldwide. The bark beetle Hypocryphalus mangiferae (Stebbing) (Coleoptera: Curculionidae: Scolytinae) has been suggested as a potential vector of the disease based primarily on field observations with little or no supporting empirical data. In this study, we investigated the role of infected mango trees in host attraction and colonization by H. mangiferae to determine if beetle attack and colonization contributes to the disease progression on mango trees. Initially, the role of various stress factors on beetle attraction and disease progression was assessed under lathe house conditions from 2008 to 2009. Results suggest that symptomatic or recently inoculated mango trees (without any obvious symptoms) are preferentially colonized by H. mangiferae. Although not significant, high numbers of beetles attacked stressed or wounded mango trees, compared to healthy or dead mango trees. Disease symptoms after beetle colonization, such as bark splitting, wilting and oozing, were further evaluated. These symptoms showed positive correlation with the degree of disease severity and host plant condition. Furthermore, two fungi, Ceratocystis fimbriata and Lasiodiplodia theobromae, were frequently isolated from the beetle and beetle-colonized trees. Based on these findings, they suggests that H. mangiferae can vector multiple fungi associated with mango sudden decline disease and play a significant role in outbreaks of this disease.     

Development of a sensitive molecular detection assay for mango malformation disease caused by <Emphasis Type="Italic">Fusarium mangiferae</Emphasis>

Objectives

To develop a sensitive and specific molecular assay for detection of mango malformation disease (MMD), which is caused primarily by Fusarium mangiferae.

Results

We screened 100 ISSR primers and identified one (UBC888) that directed the stable amplification of a specific gene fragment of 479 bp (GenBank accession number KJ526382). Based on the DNA sequence of this fragment, a pair of SCAR primers (W342 and W1772) were designed to amplify another gene fragment of 1376 bp (GenBank accession number KJ526383), demonstrating the successful conversion of an ISSR marker to a SCAR marker. An effective and simple detection assay for MMD was established based on this pair of PCR primers, with a high level of specificity and sensitivity to the DNA of F. mangiferae and other species of Fusarium both in vitro and in vivo. It can detect as little as 10 pg fungal DNA from the DNA of mango’s tissues.

Conclusions

Our assay provides a practical method for the early diagnosis so that proper prevention of the mango malformation disease can be developed.

     

Fusarium Species Associated with Mango Malformation in Peninsular Malaysia

Mango malformation has become the most important global disease on mango. Fusarium species previously associated with this disease include F. mangiferae, F. mexicanum, F. sterilihyphosum, F. proliferatum, F. subglutinans and F. tupiense. A few strains of F. proliferatum have been reported from Malaysia, but in this study, we report the results of more extensive sampling. The recovered strains were evaluated with morphology, mating tester strain cross‐fertility, amplified fragment length polymorphisms (AFLPs), and partial DNA sequences of the genes encoding translation elongation factor 1‐α (tef‐1α) and β‐tubulin (tub‐2). Amongst the 43 strains evaluated, three species were identified – F. proliferatum, F. mangiferae and F. subglutinans – with F. proliferatum being the most frequent (69%). None of the Fusarium species that appear to originate in the Americas were recovered in Malaysia, which suggests special measures may be warranted to keep these species from entering the country.     

First Occurrence of <Emphasis Type="Italic">Sternochetus mangiferae</Emphasis> (Fabricius) (Coleoptera: Curculionidae) in Brazil

Specimens of the mango stone weevil Sternochetus mangiferae (Fabricius) (Coleoptera: Curculionidae) were found in fruits of mango from a tree in the residential area of the Rio de Janeiro, RJ. This is the first report of the S. mangiferae in Brazil, currently regulated as an absent quarantine pest in the country. A taxonomist specialized in Curculionidae confirmed the identification based on morphological diagnostics characteristics. This detection is a relevant finding, because Brazil is a major producer and exporter of mango and the main areas of mango for exportation are located very far from this detection point. This pest damages seed and embryo of mango fruits and it causes reduction of fruit size and its premature dropping. The detection was notified to the Plant Health Department, division of the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), which is the National Plant Protection Organization of Brazil.     

Fused lobed anther and hooked stigma affect pollination,fertilization and fruit set in mango: A scanning electron microscopy study

Mango malformation is the most threaten disease that limits mango production, worldwide. For a long time, due to its complex nature, the cause and causal agents were strongly disputed. Diverse Fusaria, including Fusarium mangiferae, are known to be associated with the disease. There are indications that augmented level of endogenous ethylene in response to various abiotic and biotic stresses alters the morphology of reproductive organs. Here, scanning electron microscopy (SEM) of healthy and malformed reproductive organs of mango cv. Baramasi was performed to compare the functional morphology. The SEM study revealed that anthers of hermaphrodite healthy flowers were bilobed with large number of turgid pollen grains whereas malformed flowers showed fused lobed anthers with scanty deformed pollen grains. Furthermore, the stigma of healthy flowers exhibited a broad landing pad as compared to malformed stigma which showed hooked and pointed tip. All these impaired morphology of male and female reproductive organs lead to failure of sexual reproduction. This is the first evidence to show fused lobed anther with impaired pollen grains and hooked stigma with poor stigmatic receptivity are mainly responsible for restricting the pollen germination and pollen tube growth. Here we suggest that abnormal development of anthers and pistils is due to endogenously produced stress ethylene. Further, added load of cyanide, a byproduct of ethylene biosynthesis, may also contribute to the development of necrosis which lead to desiccation of anther and pistil during hypersensitive response of plants.     

Control of mango seed weevils (Sternochetus mangiferae) using the African Weaver Ant (Oecophylla longinoda Latreille) (Hymenoptera: Formicidae)

The mango seed weevil, Sternochetus mangiferae (Fabricius), is among the major threats to mango production in Tanzania. Sternochetus mangiferae is primarily a quarantine pest whose presence inside the fruits restricts access to new foreign markets and leads to rejections of fruits destined for export. Management options for the pest have largely been dependent on field sanitation and application of synthetic insecticides with some success. Thus, more sustainable methods are needed to substitute insecticides, as this may also open up opportunities for organic markets. We conducted field experiments for two fruiting seasons in a mango plantation at Mlandizi, Kibaha district, along the coastal belt of Tanzania to evaluate and compare the effectiveness of the predaceous ant Oecophylla longinoda Latreille with foliar insecticidal sprays of Dudumida (70 WDG Imidacloprid) in controlling S. mangiferae. Mango seed weevil infestation was assessed fortnightly based on infestation marks on developing fruits starting eight weeks after fruit set to early ripening phase. Between 50 and 64 fruits were sampled, well labelled in jute bags, secured and transported to the laboratory at Kibaha Biological Control Unit (KBCU) for incubation at room temperature using rearing transparent containers. Two weeks later, the fruits were dissected and inspected for the presence of S. mangiferae developmental stages. Field and laboratory results indicated that fruits from trees that were occupied by O. longinoda and from those treated with insecticide showed significantly (P < 0.0001) lower incidences and infestation rates by S. mangiferae than from untreated trees. Furthermore, there were no significant differences between the insecticide and the weaver ant treatments. We conclude that in our experiments, O. longinoda is an efficient biological control agent for a long‐term control programme and is comparable to insecticide (Dudumida) in suppressing S. mangiferae and may be used in Tanzanian mango plantations.     

First evidence of putrescine involvement in mitigating the floral malformation in mangoes: A scanning electron microscope study

Floral malformation is the most destructive disease in mangoes. To date, the etiology of this disease has not been resolved. There are indications that stress-stimulated ethylene production might be responsible for the disease. Putrescine mediates various physiological processes for normal functioning and cellular metabolism. Here, the effect of putrescine in concentration ranging from 10^?1 to 10^?3 M was evaluated on disease incidence during mango flowering seasons of 2012 and 2013. In a scanning electron microscopy (SEM) study, putrescine (10^?2 M)-treated malformed floral buds bloomed into opened flowers with separated sepals and/or petals like healthy, whereas the untreated (control) malformed buds remained deformed. Further, malformed flowers recovered upon putrescine treatment, displaying clearly bilobed anthers, enclosing a large number of normal pollen grains and functional ovary with broad stigmatic surface as compared to control. The present findings provide the first report to demonstrate the role of putrescine in reducing various adverse effects of stress ethylene via decelerating the higher pace of its biosynthesis. It stabilizes the normal morphology, development, and functions of malformed reproductive organs to facilitate successful pollination, fertilization, and, thereby, fruit set in mango flowers. However, putrescine–ethylene-mediated cell signaling network, involving various genes to trigger the response, which regulates a wide range of developmental and physiological processes leading to normal cell physiology, needs to be investigated further.     

Comparative efficacy of different pesticides against mango bark beetle Hypocryphalus mangiferae Stebbing (Coleoptera: Scolytidae)

Hypocryphalus mangiferae Stebbing is one of the most destructive insect pests of mango trees and is found to be associated with the transmission of causal organisms of mango sudden death disease. The present study was carried out to evaluate the toxicity of deltamethrin, bifenthrin, chlorpyrifos, emamectin benzoate, imidacloprid and spinosad in laboratory and field trials. Bioassay results showed that the toxicity of chlorpyrifos was significantly higher than deltamethrin but similar to bifenthrin. Deltamethrin and bifenthrin toxicity, however, increased significantly (P < 0.01) from day 1 to day 3. Spinosad was the least toxic compound while emamectin was the most toxic among new chemical insecticides tested, but its toxicity increased significantly from day 1 to day 5. Comparison of the efficacies of the insecticides using lethal times to produce 50% mortality (LT₅₀) and 90% mortality (LT₉₀) showed that the relative potencies of chlorpyrifos, emamectin, imidacloprid and spinosad were greater than bifenthrin and deltamethrin. The results of field trials showed the highest number of beetles emerged from the control twigs while significantly fewer beetles emerged from the twigs treated with bifenthrin (P < 0.05), which accounted for 12% for bifenthrin compared to that of the control. The present study demonstrated increased toxicities of systemic insecticides and chlorpyrifos compared to toxicities of deltamethrin and bifenthrin, suggesting these insecticides could be an alternative tool in a comprehensive H. mangiferae management program to eradicate the beetles from mango orchards.     

A new species of Aprostocetus Westwood (Hymenoptera: Eulophidae), parasitizing mango leaf gall midge (Diptera: Cecidomyiidae), from India

A new species of Aprostocetus doonensis Singh sp. nov. is described from northern India. The new species is a parasitoid of mango leaf gall midge, Procontarinia mangiferae (Felt) (Diptera: Cecidomyiidae). Some observations on biology and parasitization rate are also given.     

Intensive aquaculture selects for increased virulence and interference competition in bacteria

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003–2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak. Our data suggest that the farming environment may select for bacterial strains that have high virulence at both long and short time scales, and it seems that these strains have also evolved increased ability for interference competition. Our results are consistent with the suggestion that selection pressures at fish farms can cause rapid changes in pathogen populations, which are likely to have long-lasting evolutionary effects on pathogen virulence. A better understanding of these evolutionary effects will be vital in prevention and control of disease outbreaks to secure food production.     

Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.     

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.     

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.     

Development of DArT markers and assessment of diversity in Fusarium oxysporum f. sp. ciceris,wilt pathogen of chickpea (Cicer arietinum L.)

Background

Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. The severity and yield losses of Fusarium wilt differ from place to place owing to existence of physiological races among isolates. Diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. The advantages of using molecular markers to understand the distribution of genetic diversity in Foc populations is well understood. The recent development of Diversity Arrays Technology (DArT) offers new possibilities to study the diversity in pathogen population. In this study, we developed DArT markers for Foc population, analysed the genetic diversity existing within and among Foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of Foc in India.

Results

We report the successful development of DArT markers for Foc and their utility in genotyping of Foc collections representing five chickpea growing agro-ecological zones of India. The DArT arrays revealed a total 1,813 polymorphic markers with an average genotyping call rate of 91.16% and a scoring reproducibility of 100%. Cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of Foc were partially classified based on geographical source. Diversity in Foc population was compared with the phenotypic variability and it was found that DArT markers were able to group the isolates consistent with its virulence group. A number of race-specific unique and rare alleles were also detected.

Conclusion

The present study generated significant information in terms of pathogenic and genetic diversity of Foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. The DArT markers were proved to be a powerful diagnostic tool to study the genotypic diversity in Foc. The high number of DArT markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-454) contains supplementary material, which is available to authorized users.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Volatile Substances Produced by Fusarium oxysporum from Coffee Rhizosphere and Other Microbes affect Meloidogyne incognita and Arthrobotrys conoides

Microorganisms produce volatile organic compounds (VOCs) which mediate interactions with other organisms and may be the basis for the development of new methods to control plant-parasitic nematodes that damage coffee plants. In the present work, 35 fungal isolates were isolated from coffee plant rhizosphere, Meloidogyne exigua eggs and egg masses. Most of the fungal isolates belonged to the genus Fusarium and presented in vitro antagonism classified as mutual exclusion and parasitism against the nematode-predator fungus Arthrobotrys conoides (isolated from coffee roots). These results and the stronger activity of VOCs against this fungus by 12 endophytic bacteria may account for the failure of A. conoides to reduce plant-parasitic nematodes in coffee fields. VOCs from 13 fungal isolates caused more than 40% immobility to Meloidogyne incognita second stage juveniles (J₂), and those of three isolates (two Fusarium oxysporum isolates and an F. solani isolate) also led to 88-96% J₂ mortality. M. incognita J₂ infectivity decreased as a function of increased exposure time to F. oxysporum isolate 21 VOCs. Gas chromatography-mass spectrometry (GC-MS) analysis lead to the detection of 38 VOCs produced by F. oxysporum is. 21 culture. Only five were present in amounts above 1% of the total: dioctyl disulfide (it may also be 2-propyldecan-1-ol or 1-(2-hydroxyethoxy) tridecane); caryophyllene; 4-methyl-2,6-di-tert-butylphenol; and acoradiene. One of them was not identified. Volatiles toxic to nematodes make a difference among interacting microorganisms in coffee rhizosphere defining an additional attribute of a biocontrol agent against plant-parasitic nematodes.