Microorganisms Associated with the Ambrosial Beetle Xyleborus affinis with Plant Growth-Promotion Activity in Arabidopsis Seedlings and Antifungal Activity Against Phytopathogenic Fungus Fusarium sp. INECOL_BM-06.

Plants interact with a great diversity of microorganisms or insects throughout their life cycle in the environment. Plant and insect interactions are common; besides, a great variety of microorganisms associated with insects can induce pathogenic damage in the host, as mutualist phytopathogenic fungus. However, there are other microorganisms present in the insect-fungal association, whose biological/ecological activities and functions during plant interaction are unknown. In the present work evaluated, the role of microorganisms associated with Xyleborus affinis, an important beetle species within the Xyleborini tribe, is characterized by attacking many plant species, some of which are of agricultural and forestry importance. We isolated six strains of microorganisms associated with X. affinis shown as plant growth-promoting activity and altered the root system architecture independent of auxin-signaling pathway in Arabidopsis seedlings and antifungal activity against the phytopathogenic fungus Fusarium sp. INECOL_BM-06. In addition, evaluating the tripartite interaction plant-microorganism-fungus, interestingly, we found that microorganisms can induce protection against the phytopathogenic fungus Fusarium sp. INECOL_BM-06 involving the jasmonic acid-signaling pathway and independent of salicylic acid-signaling pathway. Our results showed the important role of this microorganisms during the plant- and insect-microorganism interactions, and the biological potential use of these microorganisms as novel agents of biological control in the crops of agricultural and forestry is important.

Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens.

Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.

Structural Differences in the Digestive Tract Between Females and Males Could Modulate Regurgitation Behavior in Anastrepha ludens (Diptera: Tephritidae).

With the aim of understanding the mechanisms involved in the regurgitation behavior of tephritid flies, we performed a structural study of the digestive system of the economically important fruit-fly pest, Anastrepha ludens (Loew) using optical, scanning electronic microscopy (SEM) and transmission electron microscopy (TEM), plus a feeding assay. Most structures studied are similar to those previously reported in other adult dipterans, but, importantly, we found sexual differences in some structures that apparently affect regurgitation. We report for the first time sexual differences in the crop duct nerve and large numbers of dense core vesicles within the nerve bundle. Male nerve bundles are bigger and have more secretory vesicles than female ones. The close proximity to the muscles of both the crop lobes and duct suggest that these vesicles (i.e., possibly neurosecretions) might help modulate the muscles regulating regurgitation. The salivary glands are connected to the crop via tracheae, however, SEM/TEM studies failed to find any direct structural connection. Results of the feeding assay indicate that, independently of food type (sucrose or protein) and age, males regurgitate significantly more than females. Regurgitation behavior may also play an important role in capturing bacteria in the environment, and possibly help adults eliminate ingested toxicants such as insecticides. Our findings shed light on an interesting phenomenon that has important practical implications.

[Morphological and molecular characterization of isolates of Macrophomina phaseolina associated with sugarcane in Mexico]. / Caracterización morfológica y molecular de aislados de Macrophomina phaseolina asociados a caña de azúcar en México.

Charcoal rot caused by Macrophomina phaseolina is an important disease of sugarcane in Mexico. This study was carried out to characterize isolates of M. phaseolina obtained from sugarcane by the combination of morphological and molecular analyses. The morphological characterization of 10 isolates was performed using scanning electron microscopy and light microscopy. To confirm the morphological identification, rDNA from two representative isolates was extracted, and the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction and sequenced using specific primers MpKF1 and MpKR1. Based on their morphological characteristics, all isolates were identified as M. phaseolina. Moreover, the analysis of two ITS sequences showed 100% similarity with the M. phaseolina sequences deposited in the GenBank. To our knowledge, this is the first study in the world aimed at characterizing isolates of M. phaseolina obtained from sugarcane.

New Insights on Antennal Sensilla of Anastrepha ludens (Diptera: Tephritidae) Using Advanced Microscopy Techniques.

Using light, transmission, scanning electron, and confocal microscopy, we carried out a morphological study of antennal sensilla and their ultrastructures of the Mexican Fruit Fly Anastrepha ludens (Loew), an economically important species that is a pest of mangos and citrus in Mexico and Central America. Our goal was to update the known information on the various sensilla in the antennae of A. ludens, involved in the perception of odors, temperature, humidity, and movement. Based on their external shape, size, cuticle-thickness, and presence of pores, we identified six types of sensilla with 16 subtypes (one chaetica in the pedicel, four clavate, two trichoid, four basiconic, one styloconic, and one campaniform-like in the flagellum, and three additional ones in the two chambers of the sensory pit (pit-basiconic I and II, and pit-styloconic)), some of them described for the first time in A. ludens. We also report, for the first time, two types of pores in the sensilla (hourglass and wedge shapes) that helped classify the sensilla. Additionally, we report a campaniform-like sensillum only observed by transmission electronic microscopy on the flagellum, styloconic and basiconic variants inside the sensory pit, and an "hourglass-shaped" pore in six sensilla types. We discuss and suggest the possible function of each sensillum according to their characteristics and unify previously used criteria in the only previous study on the topic.

Antifungal Effect of Copper Nanoparticles against Fusarium kuroshium, an Obligate Symbiont of Euwallacea kuroshio Ambrosia Beetle.

Copper nanoparticles (Cu-NPs) have shown great antifungal activity against phytopathogenic fungi, making them a promising and affordable alternative to conventional fungicides. In this study, we evaluated the antifungal activity of Cu-NPs against Fusarium kuroshium, the causal agent of Fusarium dieback, and this might be the first study to do so. The Cu-NPs (at different concentrations) inhibited more than 80% of F. kuroshium growth and were even more efficient than a commercial fungicide used as a positive control (cupric hydroxide). Electron microscopy studies revealed dramatic damage caused by Cu-NPs, mainly in the hyphae surface and in the characteristic form of macroconidia. This damage was visible only 3 days post inoculation with used treatments. At a molecular level, the RNA-seq study suggested that this growth inhibition and colony morphology changes are a result of a reduced ergosterol biosynthesis caused by free cytosolic copper ions. Furthermore, transcriptional responses also revealed that the low- and high-affinity copper transporter modulation and the endosomal sorting complex required for transport (ESCRT) are only a few of the distinct detoxification mechanisms that, in its conjunction, F. kuroshium uses to counteract the toxicity caused by the reduced copper ion.

Insights of the Neofusicoccum parvum-Liquidambar styraciflua Interaction and Identification of New Cysteine-Rich Proteins in Both Species.

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch's postulates-followed by a molecular approach-revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue-N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction.

Morphometrics of Amblyomma mixtum in the State of Veracruz, Mexico.

The tick Amblyomma mixtum is an ectoparasite of veterinary and public health importance because of its role as a vector of zoonotic pathogens. However, little is known about A. mixtum intraspecific variability and if morphological differentiation exists between populations across its geographic range. This study aimed to determine by electron microscopy the morphological variability of A. mixtum populations in the state of Veracruz, which has a large livestock population among states in Mexico. Forty male and 40 female A. mixtum collected from the 10 natural regions of Veracruz state were analyzed microscopically to accomplish main component analysis for each sex. Clusters were calculated with the out-tree method and a dendrogram produced to group the specimens according to their morphometric characteristics. Using 10 main components, 77% of the morphological variation of the ticks was explained. This is a reflection of scarce intraspecific variation between females. The dendogram for females grouped six clusters of specimens with similar characteristics. Morphometric variability in males was described using eight main components. Limited intraspecific variation was also observed between males. In males, the dendogram yielded six groups with similar morphometric characteristics. Morphometric analyses confirmed that the only species from the Amblyomma cajennense complex that are parasites to livestock in Veracruz state is A. mixtum. The eryxenous nature of A. mixtum combined with the frequent movement of livestock hosts may contribute to the apparent homogeneous phenotype of this tick species in Mexico.

Green-synthesized copper nanoparticles as a potential antifungal against plant pathogens.

The fabrication of fungicides in cost-effective and eco-friendly ways is particularly important for agriculture. Plant pathogenic fungi produce many economic and ecological problems worldwide, which must be controlled with potent fungicides. Here we propose the green synthesis of fungicides, which consist of copper nanoparticles (Cu-NPs) prepared in aqueous media. Through in vitro experiments, the antifungal efficacy against Fusarium solani, Neofusicoccum sp., and Fusarium oxysporum was investigated. Although the antifungal activity differs for each fungal species, it was found that the Cu-NPs induce strong morphological changes in the mycelium. Additionally, the damage of the cell membranes of the pathogens was revealed by microscopic observations. For the three evaluated fungi, fluorescence microscopy demonstrated the intracellular generation of reactive oxygen species in the mycelium. This work proves that the green-synthesized Cu-NPs are potential fungicides against F. solani, Neofusicoccum sp., and F. oxysporum.

Aqueous-phase synthesis of nanoparticles of copper/copper oxides and their antifungal effect against Fusarium oxysporum.

Different copper based-materials have been used for controlling some fungal and bacterial pathogens. However, the antifungal activity of the copper-based materials depends on different parameters, such as the crystal phase, synthesis route, and size of the particles. Herein a facile route synthesis method of Cu/CuxO-NPs was achieved through the aqueous phase. The influence of NaBH4 concentration on the phase composition was studied. The synthesized Cu/CuxO-NPs were characterized by X-Ray diffraction (XRD), Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering. Five Cu/CuxO-NPs with different phase composition and nanoparticle size were obtained. The antifungal activity of the synthesized Cu/CuxO-NPs was studied in vitro against Fusarium oxysporum. The results indicate that a high percent of inhibition of radial growth (IGR) was obtained with NPs, which have a higher proportion of Cu2O phase and relatively smaller size particles. Furthermore, hypha morphology, membrane damage and production of reactive oxygen species (ROS) was evaluated with SEM and confocal microscopy.

Occurrence of Amblyomma mixtum on the water buffalo (Bubalus bubalis) in Mexico.

The water buffalo (Bubalus bubalis) is an Asian species of bovine that was introduced in Mexico in 1992 as an alternative for milk and meat production. To date, no surveys have been conducted to identify ticks acquired by water buffaloes since their arrival in the country. Here we report, for the first time, the presence of Amblyomma mixtum, a neotropical tick in the Amblyomma cajennense complex, found on water buffaloes from Mexico and discuss its possible implications in veterinary public health for the region.

Leaf Epiphytic Bacteria of Plants Colonizing Mine Residues: Possible Exploitation for Remediation of Air Pollutants.

Plant surfaces are known as an important sink for various air pollutants, including particulate matter and its associated potentially toxic elements (PTE). Moreover, leaves surface or phylloplane is a habitat that harbors diverse bacterial communities (epiphytic). However, little is known about their possible functions during phytoremediation of air pollutants like PTE. The study of leaf epiphytic bacteria of plants colonizing mine residues (MR) containing PTE is thus a key to understand and exploit plant-epiphytic bacteria interactions for air phytoremediation purposes. In this research, we aimed (i) to characterize the functions of epiphytic bacteria isolated from the phylloplane of Brickellia veronicifolia, Flaveria trinervia, Gnaphalium sp., and Allionia choisyi growing spontaneously on multi-PTE contaminated MR and (ii) to compare these against the same plant species in a non-polluted control site (NC). Concentrations (mg kg-1) of PTE on MR leaf surfaces of A. choisyi reached up to 232 for Pb, 13 for Cd, 2,728 for As, 52 for Sb, 123 for Cu in F. trinervia, and 269 for Zn in Gnaphalium sp. In the four plant species, the amount of colony-forming units per cm2 was superior in MR leaves than in NC ones, being A. choisyi the plant species with the highest value. Moreover, the proportion of isolates tolerant to PTE (Zn, Cu, Cd, and Sb), UV light, and drought was higher in MR leaves than in those in NC. Strain BA15, isolated from MR B. veronicifolia, tolerated 150 mg Zn L-1, 30 mg Sb L-1, 25 mg Cu L-1; 80 mg Pb L-1, and was able to grow after 12 h of continuous exposition to UV light and 8 weeks of drought. Plant growth promotion related traits [N fixation, indole acetic acid (IAA) production, and phosphate solubilization] of bacterial isolates varied among plant species isolates and between MR and NC sampling condition. The studied epiphytic isolates possess functions interesting for phytoremediation of air pollutants. The results of this research may contribute to the development of novel and more efficient inoculants for microbe-assisted phytoremediation applied to improve air quality in areas exposed to the dispersion of metal mine tailings.

In Vitro Lethal Activity of the Nematophagous Fungus Clonostachys rosea (Ascomycota: Hypocreales) against Nematodes of Five Different Taxa.

This study was aimed to evaluate the in vitro lethal activity of the nematophagous fungi Clonostachys rosea against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained C. rosea cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: Haemonchus contortus, Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus, and Butlerius sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: H. contortus (L3) 87.7%; C. elegans 94.7%; Rhabditis sp. 71.9%; P. redivivus 92.7%; and Butlerius sp. 100% (p ≤ 0.05). The activity showed by C. rosea against the H. contortus can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.

Phytobarriers: Plants capture particles containing potentially toxic elements originating from mine tailings in semiarid regions.

Retention of particles containing potentially toxic elements (PTEs) on plants that spontaneously colonize mine tailings was studied through comparison of washed and unwashed shoot samples. Zn, Pb, Cd, Cu, Ni, Co and Mn concentrations were determined in plant samples. Particles retained on leaves were examined by Scanning Electronic Microscopy and energy dispersive X-Ray analysis. Particles containing PTEs were detected on both washed and unwashed leaves. This indicates that the thorough washing procedure did not remove all the particles containing PTEs from the leaf surface, leading to an overestimation of the concentrations of PTEs in plant tissues. Particularly trichomes and fungal mycelium were retaining particles. The quantity and composition of particles varied among plant species and place of collection. It is obvious that plants growing on toxic mine tailings form a physical barrier against particle dispersion and hence limit the spread of PTEs by wind.

Caracterización morfológica y molecular de aislados de Macrophomina phaseolina asociados a caña de azúcar en México / Morphological and molecular characterization of isolates of Macrophomina phaseolina associated with sugarcane in Mexico

La pudrición carbonosa, causada por Macrophomina phaseolina, es una enfermedad importante de la caña de azúcar en México. Este estudio se realizó con el objetivo de caracterizar aislados de M. phaseolina obtenidos de caña de azúcar mediante análisis morfológicos y moleculares. La caracterización morfológica de 10 aislados se llevó a cabo con el uso de microscopia electrónica de barrido y microscopia de luz. Para confirmar la identificación, se extrajo el ADNr de 2 aislados representativos, y la región del espaciador interno transcrito (ITS) se amplificó mediante la reacción en cadena de la polimerasa y se secuenció usando los iniciadores específicos MpKF1 y MpKR1. Los aislados se identificaron como M. phaseolina con base en la morfología. El análisis de secuencias ITS mostró 100% de similitud con las secuencias de M. phaseolina depositadas en el GenBank. Para nuestro conocimiento, este es el primer estudio del mundo enfocado a caracterizar aislados de M. phaseolina obtenidos de caña de azúcar

Charcoal rot caused by Macrophomina phaseolina is an important disease of sugarcane in Mexico. This study was carried out to characterize isolates of M. phaseolina obtained from sugarcane by the combination of morphological and molecular analyses. The morphological characterization of 10 isolates was performed using scanning electron microscopy and light microscopy. To confirm the morphological identification, rDNA from two representative isolates was extracted, and the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction and sequenced using specific primers MpKF1 and MpKR1. Based on their morphological characteristics, all isolates were identified as M. phaseolina. Moreover, the analysis of two ITS sequences showed 100% similarity with the M. phaseolina sequences deposited in the GenBank. To our knowledge, this is the first study in the world aimed at characterizing isolates of M. phaseolina obtained from sugarcane