First report of Fusarium equiseti causing root and crown rot in tomato in Mexico.

Tomato (Solanum lycopersicum L.) is one of the most important crops in Mexico due to its economic and nutritional value. Among the main diseases in tomato production is Fusarium wilt, which can cause 60% production losses (Ascencio et al, 2008). Mixed infections of Fusarium species or other fungi genera, would increase disease severity. During April to May of 2021, tomato plants with more than 60 days old, were collected from the main production areas of Aguascalientes (22°03'46.5"N 102°05'17.4"W and 22°04'53.64"N 101°58'55.81"W) and Zacatecas (23°05'59.2"N 102°41'07.3"W and 22°16'52.1"N 102°00'11.8"W) Mexico states. Plants showed main root rot, vascular bundles necrosis with corky appearance, stem crown rot, and ascending yellowing. The main root and stem crown were cut in 0.25 cm2 pieces and disinfested in 2% NaClO for one minute, rinsed with distilled water two times, placed on acidified potato dextrose agar (PDA) medium, and incubated at 25 ± 2°C for 7 days. Characteristic Fusarium growths were purified by hyphal tip on PDA, subsequently pure strains were obtained by single-spore isolation method. Several fungi colonies were obtained, but we focused on the colonies that showed abundant aerial mycelium of white color and irregular growth, which turned yellowish to golden and brown color as it ages. Carnation leaf agar (CLA) medium were used for conidia and sporodochium development. Chains of terminal, intercalary and agglomerated chlamydospores with thick, rough brown walls of 18.9 (7.46) µm in diameter (n=120) were observed in the mycelium. Macroconidia with 5 to 7 septa were 30 to 75 (28.32) µm in long and 1.2 to 4.8 (3.2) µm in wide (n=72). Basal cell developed in foot-shape, apical cell was elongated and slightly curved, and some macroconidia had swollen midd-cell. Sporodochium was orange to brown in color and microconidia were absent (Figure 1). Two representative strains from each state, LCA-3.1 and EMA-1 from Aguascalientes and ECZ-4 and LRZ-6 from Zacatecas, were selected for DNA amplification of ITS, TEF-1α and RPB2 regions, with universal primers ITS1/ITS4, EF1/EF2 and 2-5F2/7cR (White et al.1990; O'Donnell et al. 1998, 2013). PCR products were sequenced by Psomagen, Inc. (USA). The sequences obtained showed 100% of similarity among themselves and within species of the Fusarium incarnatum-equiseti species complex (FIESC) with nucleotide NCBI accessions NR_121457 (Type material) for ITS and MW362069 for TEF-1α; and 99.28% with MN170399 for RPB2 in FUSARIOID-ID database. According to morphological (Leslie and Summerell, 2006) and molecular characteristics, isolates were identified as Fusarium equiseti (FIESC 14). The LCA-3.1 sequences were selected to be deposited in GenBank with accession numbers OM812801 (ITS), OM937108 (TEF-1α) and ON653596 (RPB2). Pathogenicity tests were performed twice, under greenhouse conditions in tomato seedlings of cv. Rio Grande. Five tomato seedlings were inoculated by root immersion method (Lopez et al, 2018) in a 1x106 spores/mL solution for 8 min, and transplanted to 1L pots with sterile peat. Five controls plants were immersed in sterile water. At 14 days after inoculation, a general plant decline and slower growth compared to the control plants were observed. Subsequently, plants showed root rot, vascular necrosis, and a brown ring in stem crown. Controls were symptomless. The fungi were re-isolated from symptomatic plants and were morphologically similar to the inoculated strains. Patel et al. (2017) described the pathogenic and toxic effects of F. equiseti on tomato, causing low seed germination, and low root and shoot growth. This is the first report of F. equiseti causing root and stem rot in tomato plants in Mexico.

Aedes albopictus in northeast Mexico: an update on adult distribution and first report of parasitism by Ascogregarina taiwanensis.

BACKGROUND & OBJECTIVES: Aedes albopictus has been known as efficient vector of dengue in Asian countries and its wide displacement of Ae. aegypti has been documented in many parts of the world. The present survey was carried out to update the distribution of Ae. albopictus in northeast Mexico and to report the first record of parasitism of mosquitoes by Ascogregarina taiwanensis in Mexico. METHODS: Human landing collections were conducted in the month of May every year between 2007 and 2009 across the three states, Nuevo Leon (NL), Tamaulipas and Coahuila in northeast Mexico. Six human bait collections were also organized at the cemetery of Gomez Farias (GF), a village in southern Tamaulipas during the rainy and dry seasons in 2010. Aedes albopictus caught in 2010 were dissected for parasitic protozoan gregarines. RESULTS: The results of human landing collections carried out during 2007-10 across the three states of northeast zone of Mexico revealed that Ae. albopictus is invading along the route between Monterrey City in NL and Tampico, Tamaulipas, but not into the arid state of Coahuila. Aedes albopictus was recorded in nine new municipalities in addition to the 15 municipalities reported before 2005. Furthermore, six human-bait collections performed during the dry and rainy seasons in 2010 at the cemetery of GF suggest the exclusion of Ae. aegypti on that site. Dominance was shared by Ae. quadrivittatus, another container-inhabitant but indigenous species, and Ae. albopictus during the dry and rainy seasons, respectively. The results of dissection of the mosquitoes for gregarines revealed the parasitism of Ae. albopictus by A. taiwanensis. INTERPRETATION & CONCLUSION: The results of this study showed that Ae. albopictus has spread to all the municipalities in the northeastern Mexico except the arid area and reported the first record of parasitic protozoan A. taiwanensis in Mexico. We recommend further studies on larval and adult populations of natural container-occupant mosquitoes in northeastern Mexico in order to have a better documentation of the impact of Ae. albopictus upon the indigenous species community, and its epidemiological role in dengue transmission.

Gastrointestinal parasites in bullfrogs (Lithobates catesbeianus) in aquaculture production units in the Mexican central highlands.

In Mexico, intensive production of bullfrogs is one of the most important aquaculture activities, due to growing demand for their meat. Frogs can be hosts for several parasites that negatively affect their development and health. The objective of this study was to identify the presence of intestinal parasites in bullfrogs in aquaculture production units. Eighteen bullfrogs aquaculture production units were selected, and 20 animals (n=360) from each farm. Fecal samples were obtained by mucosal scraping and processed using the concentration method. The overall prevalence of intestinal parasites was 70.5%, and all farms had frogs infected by some species of parasite. Two species of parasites were identified: Eimeria sp. and Strongyloides sp. Significant differences were found regarding parasite prevalence between males and females (73.8% vs 58.8%) and regarding tibia length (5.5 vs 6.1 cm) and weight (168 vs 187 g) between parasitized and non-parasitized frogs. In conclusion, the present study showed a high prevalence of intestinal parasites, and morphometric alterations (weight, snout-cloaca length, radio-ulna length, tibia length and distance between parotid glands) were identified in the parasitized animals. These results provided useful information that will enable establishment of adequate control measures to help minimize the adverse effects of these parasites.

Gastrointestinal parasites in bullfrogs (Lithobates catesbeianus) in aquaculture production units in the Mexican central highlands / Parasitos gastrointestinais em rãs-touro (Lithobates catesbeianus) em unidades de produção aquícola do planalto central do México

In Mexico, intensive production of bullfrogs is one of the most important aquaculture activities, due to growing demand for their meat. Frogs can be hosts for several parasites that negatively affect their development and health. The objective of this study was to identify the presence of intestinal parasites in bullfrogs in aquaculture production units. Eighteen bullfrogs aquaculture production units were selected, and 20 animals (n=360) from each farm. Fecal samples were obtained by mucosal scraping and processed using the concentration method. The overall prevalence of intestinal parasites was 70.5%, and all farms had frogs infected by some species of parasite. Two species of parasites were identified: Eimeria sp. and Strongyloides sp. Significant differences were found regarding parasite prevalence between males and females (73.8% vs 58.8%) and regarding tibia length (5.5 vs 6.1 cm) and weight (168 vs 187 g) between parasitized and non-parasitized frogs. In conclusion, the present study showed a high prevalence of intestinal parasites, and morphometric alterations (weight, snout-cloaca length, radio-ulna length, tibia length and distance between parotid glands) were identified in the parasitized animals. These results provided useful information that will enable establishment of adequate control measures to help minimize the adverse effects of these parasites.(AU)

No México, a produção intensiva de rãs-touro é uma das atividades mais relevantes da aquicultura devido à sua crescente demanda. As rãs podem ser hospedeiras de vários parasitos que afetam negativamente seu desenvolvimento e sua saúde animal. O objetivo deste trabalho é identificar a presença de parasitas gastrointestinais (IPs) em rãs de boi em unidades de produção aquícola. Foram selecionadas 18 unidades de produção aquícola de rãs-touro, e de cada unidade 20 animais (n=360). Amostras fecais foram obtidas por raspagem de mucosas e processadas pelo método de concentração. A prevalência geral de IPs foi de 70,5%, todas as fazendas foram infectadas com algum parasita. Duas espécies de parasitas Eimeria sp. e Strongyloides sp. foram identificadas; encontrando-se diferenças significativas na prevalência entre machos e fêmeas (73,8% vs 58,8%), comprimento da tíbia (5,5 vs 6,1 cm) e peso (168 vs 187 g) entre rãs parasitadas e não parasitadas. Em conclusão, o presente estudo mostrou uma alta prevalência e alterações morfométricas (peso, comprimento do ronco-cloaca, comprimento do rádio-ulna, comprimento da tíbia e distância entre as glândulas parótidas) todas identificadas nos animais parasitados. Esses resultados mostram informações relevantes que permitirão o estabelecimento de medidas de controle adequadas para ajudar a minimizar os efeitos adversos desses parasitas.(AU)

Copulation Activity, Sperm Production and Conidia Transfer in Aedes aegypti Males Contaminated by Metarhizium anisopliae: A Biological Control Prospect.

BACKGROUND: Dengue is the most prevalent arboviral disease transmitted by Aedes aegypti worldwide, whose chemical control is difficult, expensive, and of inconsistent efficacy. Releases of Metarhizium anisopliae--exposed Ae. aegypti males to disseminate conidia among female mosquitoes by mating represents a promising biological control approach against this important vector. A better understanding of fungus virulence and impact on reproductive parameters of Ae. aegypti, is need before testing auto-dissemination strategies. METHODOLOGY/PRINCIPAL FINDINGS: Mortality, mating competitiveness, sperm production, and the capacity to auto-disseminate the fungus to females up to the 5 th copulation, were compared between Aedes aegypti males exposed to 5.96 x 10(7) conidia per cm2 of M. anisopliae and uninfected males. Half (50%) of fungus-exposed males (FEMs) died within the first 4 days post-exposure (PE). FEMs required 34% more time to successively copulate with 5 females (165 ± 3 minutes) than uninfected males (109 ± 3 minutes). Additionally, fungus infection reduced the sperm production by 87% at 5 days PE. Some beneficial impacts were observed, FEMs were able to successfully compete with uninfected males in cages, inseminating an equivalent number of females (about 25%). Under semi-field conditions, the ability of FEMs to search for and inseminate females was also equivalent to uninfected males (both inseminating about 40% females); but for the remaining females that were not inseminated, evidence of tarsal contact (transfer of fluorescent dust) was significantly greater in FEMs compared to controls. The estimated conidia load of a female exposed on the 5th copulation was 5,200 mL(-1) which was sufficient to cause mortality. CONCLUSION/SIGNIFICANCE: Our study is the first to demonstrate auto-dissemination of M. anisopliae through transfer of fungus from males to female Ae. aegypti during mating under semi-field conditions. Our results suggest that auto-dissemination studies using releases of FEMs inside households could successfully infect wild Ae. aegypti females, providing another viable biological control tool for this important the dengue vector.

Hongos entomopatógenos (mycota: deuteromycetes) aislados en el Noreste de México: impacto sobre la longevidad, fecundidad, fertilidad y tasas de cópula e inseminación en aedes aegypti (diptera-culicidae)

La resistencia a los insecticidas químicos ocasionada por los insectos, ha llevado al interés en los hongos como agentes de control biológico de mosquitos. La mayoría de los trabajos con entomopatógenos se han evaluado en relación a larvas, pero hace poco se han comenzado a evaluar infecciones de mosquitos adultos a través de la exposición de superficies tratadas con conidias de hongos. En el presente estudio se evaluaron cepas mexicanas de Beauveria bassiana y Metarhizium anisopliae contra Aedes aegypti, lo que demuestra que el comportamiento de los machos impregnados con los hongos causan una mortalidad y daños en las variables de fecundidad en hembras. Todas las cepas a 6x108 conidias/ml -1 fueron altamente virulentas con una TL50 de 2.70(±0,29)-5.33(±0.53) días estimado en la exposición de hembras. Machos vírgenes de Aedes aegypti (Linneo) fueron expuestos durante 48h a la dosis de 6x108 conidias ml-1 de un aislado de Metarhizium anisopliae y luego fueron confinados con hembras vírgenes para examinar transmisión por conducta de cópula. En hembras expuestas a un macho contaminado con el aislado H-M5 hubo un 90% de mortalidad, una vida media de siete días, y fecundidad media de 0.52±0.32 huevos por hembra, la cual fue 99% más baja que la fecundidad en el control (35.35±8.3). Menos del 20% de las hembras infectadas resultaron inseminadas lo cual sugiere que la mayoría se infectaron por intentos de cópula. De la misma forma la transmisión de dos cepas de B. bassiana (H-B2 y H-B4), probadas en el apareamiento, ambos mataron entre 80-90% de las hembras en 15 días confinados con machos previamente expuestos durante 48 horas a los hongos. De las tasas de mortalidad, 27 y 48% respectivamente, fueron las infecciones adquiridas por cópulas, donde se produjo la inseminación. La TL50 para las hembras infectadas sexualmente fueron de 7.92(±0.46) y 8.82(±0.45) días para ambas cepas, mientras que el control fue de 13.92 (± 0.58). Del mismo modo, la fecundidad disminuyó entre 95% y 60% con ambos aislados en comparación con el control. El aislado H-M5 es un candidato prometedor para biocontrol vectorial del dengue, pero antes se debe estudiar el riesgo que el hongo implica para la salud pública. Para B. bassiana este es el primer reporte que se da contra Ae. aegypti a través de comportamiento de cópula de machos vírgenes hacia hembras.(AU)

Transmission of Beauveria bassiana from male to female Aedes aegypti mosquitoes.

BACKGROUND: Resistance to chemical insecticides plus high morbidity rates have lead to rising interest in fungi as candidates for biocontrol agents of mosquito vectors. In most studies fungal infections have been induced by exposure of mosquitoes to various surfaces treated with conidia. In the present study eight Mexican strains of Beauveria bassiana were assessed against Aedes aegypti by direct exposure of females to 6 × 10(8) conidia ml (-1) on a filter paper, afterwards, the transmission of the least and most virulent isolates was evaluated by mating behavior from virgin, fungus-contaminated male to females, to examine this ethological pattern as a new approach to deliver conidia against the dengue vector. METHODS: In an exposure chamber with a filter paper impregnated with 6 × 10(8) conidia ml (-1) of the least and most virulent strains of B. bassiana, 6-8 day old males of A. aegypti were exposed for 48 hours, and then transferred individually (each one was a replicate) to another chamber and confined with twenty healthy females of the same age. Clean males were used in controls. Survival, infection by true mating (insemination) or by mating attempts (no insemination) and fecundity were daily registered until the death of last female. Data analysis was conducted with proc glm for unbalanced experiments and means were separated with the Ryan test with SAS. RESULTS: All strains were highly virulent with LT(50) ranging from 2.70 (± 0.29) to 5.33 (± 0.53) days. However the most (Bb-CBG2) and least virulent (Bb-CBG4) isolates were also transmitted by mating behavior; both killed 78-90% of females in 15 days after being confined with males that had previously been exposed for 48 hours to fungi. Of these mortality rates, 23 and 38% respectively, were infections acquired by copulations where insemination occurred. The LT(50) for sexually-infected females were 7.92 (± 0.46) and 8.82 (± 0.45) days for both strains, while the one in control was 13.92 (± 0.58). Likewise, fecundity decreased by 95% and 60% for both Bb-CBG2 and Bb-CBG4 isolates in comparison with control. The role of mating attempts in this delivery procedure of B. bassiana is discussed. CONCLUSIONS: This is the first report about transmission of B. bassiana by mating behavior from virgin, fungus-contaminated males to females in A. aegypti. Fungal infections acquired by this route (autodissemination) infringed high mortality rates (90%) in mated or approached females. However, prior to releasing virgin, fungus-contaminated males to spread B. basasiana among females of A. aegypti, this novel alternative needs further investigations.

Dissemination of Metarhizium anisopliae of low and high virulence by mating behavior in Aedes aegypti.

BACKGROUND: Dengue is a viral disease transmitted by Aedes mosquitoes. It is a threat for public health worldwide and its primary vector Aedes aegypti is becoming resistant to chemical insecticides. These factors have encouraged studies to evaluate entomopathogenic fungi against the vector. Here we evaluated mortality, infection, insemination and fecundity rates in A. aegypti females after infection by autodissemination with two Mexican strains of Metarhizium anisopliae. METHODS: Two M. anisopliae strains were tested: The Ma-CBG-1 least virulent (lv), and the Ma-CBG-2 highly virulent (hv) strain. The lv was tested as non mosquito-passed (NMP), and mosquito-passed (MP), while the hv was examined only as MP version, therefore including the control four treatments were used. In the first bioassay virulence of fungal strains towards female mosquitoes was determined by indirect exposure for 48 hours to conidia-impregnated paper. In the second bioassay autodissemination of fungal conidia from fungus-contaminated males to females was evaluated. Daily mortality allowed computation of survival curves and calculation of the LT50 by the Kaplan-Meier model. All combinations of fungal sporulation and mating insemination across the four treatments were analyzed by χ2. The mean fecundity was analyzed by ANOVA and means contrasted with the Ryan test. RESULTS: Indirect exposure to conidia allowed a faster rate of mortality, but exposure to a fungal-contaminated male was also an effective method of infecting female mosquitoes. All females confined with the hv strain-contaminated male died in fifteen days with a LT50 of 7.57 (± 0.45) where the control was 24.82 (± 0.92). For the lv strain, it was possible to increase fungal virulence by passing the strain through mosquitoes. 85% of females exposed to hv-contaminated males became infected and of them just 10% were inseminated; control insemination was 46%. The hv strain reduced fecundity by up to 99%, and the lv strain caused a 40% reduction in fecundity. CONCLUSIONS: The hv isolate infringed a high mortality, allowed a low rate of insemination, and reduced fecundity to nearly zero in females confined with a fungus-contaminated male. This pathogenic impact exerted through sexual transmission makes the hv strain of M. anisopliae worthy of further research.