Nolberto Arismendi

PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries

The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples.

Absence of Transovarial Transmission of ‘Candidatus Phytoplasma ulmi’ in the Vector Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae): Is It a Rule More Than an Exception?

Abstract‘Candidatus Phytoplasma ulmi’ (Elm yellows, 16SrV-A), transmitted by Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae), has been found in native Chilean plants, and transovarial transmission has been considered as a possible form of transmission. An analysis to detect the presence of ‘Ca. Phytoplasma ulmi’ and other phytoplasmas in A. curtulus eggs, nymphs of the first and fifth instars were carried out in two experiments using nested PCR and DNA sequencing. The first experiment showed the natural acquisition of phytoplasma by adult females, and the second demonstrated the acquisition of phytoplasma in controlled conditions. Results showed that eggs and the first and fifth instars were not positive for phytoplasmas in nested PCR. ‘Candidatus Phytoplasma ulmi’ was detected and identified on average 10 and 47% of the adult females used in experiments 1 and 2, respectively. Other phytoplasma (X-disease group) was also found in adult females used in the experiment 1. We demonstrate that although gravid females contain phytoplasmas, they are not able to transmit them to their progeny, confirming that transovarial transmission of ‘Ca. Phytoplasma ulmi’ does not occur in A. curtulus.

Distribution and prevalence of crown rot pathogens affecting wheat crops in southern Chile

Crown rot pathogens are associated with higher losses for wheat crop farmers, but information about the distribution and prevalence of these pathogens in Chile is inadequate. Distribution and prevalence of wheat (Triticum aestivum L.) crown rot pathogens were examined in a survey of 48 commercial fields from December 2011 to February 2012 in southern Chile. These fields were located between Collipulli (37o56’00” S; 72°26’39” W) and Purranque (40o50’30” S; 73°22’03” W). Severity of crown rot disease was determined through visual assessment of the first internode of 20 tillers obtained from each field. Incidence of crown rot pathogens per field was determined by plating the 20 tillers on Petri plates with 20% potato dextrose agar amended with lactic acid (aPDA) medium. Resulting fungal colonies from monoxenic culture were identified by morphological or molecular-assisted identification. Severity of crown rot varied between 11.3% and 80% for individual fields. Culture plate analysis showed 72.2% of stems were infected with some fungus. Fusarium avenaceum, F. graminearum, and F. culmorum, pathogens associated with Fusarium crown rot disease were isolated from 13.5% of tillers. Gaeumannomyces graminis, causal agent of take-all disease in cereals, was isolated from 11.1% of culms. Phaeosphaeria sp., an endophyte and possibly a non-pathogenic fungus, was isolated from 13.9% of tillers. Pathogenic fungi such as Rhizoctonia spp. and Microdochium nivale, other saprophyte, and several unidentified non-sporulating fungi were isolated at frequencies lower than 3% of the total. Fusarium crown rot and take-all were the most prevalent and distributed crown rot diseases present in wheat crops in southern Chile.

Survival, fecundity, and body mass of Amplicephalus curtulus influenced by ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) infection

The leafhopper Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) can transmit ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) from a native Chilean shrub, Ugni molinae Turcz. (Myrtaceae), to ryegrasses. A recent study showed that this phytoplasma reduced the total protein content and the activity of detoxifying enzymes in A. curtulus, which could also affect its vector fitness. This study evaluated the effect of ‘Ca. Phytoplasma ulmi’ on the longevity, fecundity, and body mass of A. curtulus. Both females and males were exposed to ‘Ca. Phytoplasma ulmi’‐infected plants for 96 h, whereas a control group remained unexposed. Quartiles from adult emergence to 75% (t75), 50% (t50), and 25% (t25) survival rates were determined for each leafhopper survival distribution. The dry weight was also established at the end of the experiment. The adult lifespan of phytoplasma‐infected males and females was significantly lower than that of the uninfected leafhoppers in quartile survival distributions t50 and t25. The phytoplasma‐infected males and females lived 3 and 4 weeks less than uninfected ones in the last quartile, respectively. Fecundity was established by number of nymphs per female (in four periods) in phytoplasma‐infected and uninfected assays. In general, the weekly pattern of the number of nymphs per phytoplasma‐infected female was lower than that of uninfected leafhoppers; it was 37% lower at the end of the experiment. Phytoplasma‐infected females weighed significantly less (11%) than uninfected individuals. Phytoplasma‐infected males weighed 8% less than uninfected ones, but this difference was not significant. Our data indicated that ‘Ca. Phytoplasma ulmi’ negatively affected the fitness of A. curtulus, and nymphs produced by phytoplasma‐infected females varied over time, which may influence the disease dynamics in nature or in field crops.

Infection of ‘Candidatus Phytoplasma ulmi’ reduces the protein content and alters the activity of detoxifying enzymes in Amplicephalus curtulus

It has been reported that insecticide‐detoxifying enzymes such as glutathione S‐transferases (GST) and esterases are affected by microbial infections in hemipteran insect vectors. The total protein content, and GST and α‐ and β‐esterase activities were quantified in ‘Candidatus Phytoplasma ulmi’‐infected and uninfected adults of Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) at 25, 35, and 45 days after the acquisition access period (AAP) in the head‐thorax and abdomen sections. The total protein content was lower in phytoplasma‐infected leafhoppers 25, 35, and 45 days after the AAP. Thirty‐five days after the AAP, the GST and β‐esterase activities had increased (26 and 69%, respectively) compared to the control. However, 45 days after the AAP, the phytoplasma‐infected leafhoppers displayed lower GST (87%) and β‐esterase (253%) activities than the uninfected individuals. On the other hand, the α‐esterase activity proved to be unaffected by the phytoplasma infection. Forty‐five days after the AAP, females had a higher phytoplasma titer (46%) in their head‐thorax than in their abdomen sections, whereas males showed a higher titer in their abdomens (75%). In addition, the GST and β‐esterase activities in the abdomen were affected negatively by 96–98% as a result of the increasing ‘Ca. Phytoplasma ulmi’ titer. These results indicate that an infection of ‘Ca. Phytoplasma ulmi’ alters the metabolic activities of A. curtulus.

Evaluación del color y la posición de trampas en la captura de cicadélidos en Gaultheria phillyreifolia (Ericaceae) afectadas por fitoplasmas

Color sticky traps are one of the main alternatives to collect insect vectors, as they are easy to handle and are inexpensive. We aimed to compare the effect of color and height of the traps on the attractiveness to potential cicadellids vectors of plant pathogens. Yellow and green colored stick traps were placed at two different heights in plant of Gaultheria phillyreifolia. Seventeen leafhopper species were identified, with Ribautiana tenerrima Herrich-Shäffer (49%), Carelmapu ramosi Linnavuori & DeLong (33%), Carelmapu aurionitens Linnavuori (5%) and Atanus sp. (6%) being the most common. All these species were significantly attracted by yellow sticky traps. Ribautiana tenerrima was the only species affected by the height of the sticky traps. However, this was also dependent on the sampling season. The phytoplasma vector candidate, C. ramosi, showed two population peaks in early and late summer, which may indicate two different generations. Males of this species were more abundant than females on sticky traps, but were both similarly attracted to yellow sticky traps. No differences were detected in the capture efficiency of both sexes at any height of the traps. The high proportion of C. ramosi captured suggests that the yellow sticky traps can be an important element for monitoring this species.

Promising antimicrobial activity against the honey bee parasite Nosema ceranae by methanolic extracts from Chilean native plants and propolis

The gut parasite Nosema ceranae is one of the most prevalent honey bee microsporidia worldwide and its chemical management in American countries is mainly based on the antibiotic fumagillin. However, it has recently been reported that this drug has negative side effects in honey bees; therefore, non-harmful alternative treatments must be found. The effects of methanolic extracts (2, 4, 8 and 16%) from Chilean native plant leaves (Aristotelia chilensis, Ugni molinae, and Gevuina avellana) and propolis (Biobío (BB) and Los Ríos (LR) regions) on N. ceranae load, diet consumption and the survival of N. ceranae-infected honey bees were evaluated through oral intake. When bees were N. ceranae-infected and treated with extracts of A. chilensis (8%), U. molinae (2 and 8%) and propolis BB (8%) simultaneously, the N. ceranae load decreased significantly and the survival of infected bees also improved. On the other hand, when bees were first extracts-treated and then N. ceranae-infected, their diet consumption increased and all extracts significantly decreased the N. ceranae load in infected bees. However, only extracts from U. molinae (2 and 8%) and propolis LR (8%) maintained a higher survival rate in infected bees. Natural compounds were also identified; U. molinae and A. chilensis leaves had high concentrations of rutin and myricetin, whereas galangin and pinocembrin were found mostly in southern Chilean propolis (LR). This study constitutes the first report of the antiparasitic activity of propolis against N. ceranae in Apis mellifera and the antimicrobial activity of Chilean plant extracts against this honey bee parasite.

A scientific note on first detection of Kashmir bee virus in Apis mellifera (Hymenoptera: Apidae) in South America

Laboratories of Virology and Bee Pathology, Faculty of Agronomy, Universidad de Concepción, Av. Vicente Méndez 595, Chillán, Chile Departament of Plant Production, Faculty of Agronomy, Universidad de Concepción, Av. Vicente Méndez 595, Chillán, Chile Faculty of Bioscience Engineering, University of Ghent, Coupure Link 653, 9000, Ghent, Belgium Centro de Producción de Insectos Benéficos BioBichos Ltda, Variante Cato s/n, Chillán, Chile