First Record of Ascosphaera apis Isolated from Commercial Pollen from Northern Argentinian Provinces

Commercial pollen was obtained dried in 2014, 2015 and 2016; one random sample from each province was acquired from herbalists from the Argentinian provinces of Buenos Aires, Corrientes, Entre Ríos, Jujuy, Misiones, San Luis, Santa Fe and Tucumán (Fig. 1). Although the treatment that each sample received is not known, the norms for commercialization proposed by the National Administration of Drugs, Food and Medical Technology had to be followed, which guarantees that Argentinian products are effective, safe and of good quality. The eight samples received from herbalists were stored at 20ºC in the dark until they were processed in the laboratory. Then, 1 g of each pollen sample was seeded on a selective medium containing agar malt and yeast extracts with 20% glucose (hereafter MY20) (Takatori & Tanaka, 1982) and incubated under microaerophilia conditions at 30±2°C for fourteen days.

Fig. 1

The fungal strains were selected according to their macroscopic characteristics in the MY20 culture medium. The shape, size and color of the colony as well as the microscopic features, namely shape and size of sporocysts, asci, and ascospores (µm) were taken into account. The samples were prepared with lactophenol blue and observed at 40X and 100X under a Zeiss microscope; 1000 sporoscysts and the average size of asci and ascospores were measured (µm).

Genomic DNA was obtained from the collected samples, using the mycelium grown in MY20 liquid medium. The mycelium was washed twice with 1 mL of cold solution (Tris-HCl 0.1M, EDTA 0.02M) and spun at 12,000 rpm for 3 min. Subsequently, the mycelium was crushed with a sterile glass rod in the presence of 1 mL of extraction buffer (Tris-HCl 100mM pH 8, NaCl 1.5M, EDTA 50mM pH 8, Proteinase K, 0.1mg/ml, β-mercaptoethanol 10 mM, SDS 2%) and digested at 60ºC by agitation with vortex every 10 min. The DNA was purified from the supernatant of this digestion using chloroform:isoamyl alcohol (24:1) twice and potassium acetate 3M only once. The DNA was subsequently precipitated with 100% isopropanol, 70% cold ethanol wash, spun at 12,000 rpm for 5 min, dried at room temperature, resuspended in distilled nuclease-free water and stored at −20ºC until use. Genomic DNA was revealed in agarose gel at 1%, with 3 μL of ethidium bromide (0.008 mg/μL) for every 75 mL of agarose solution.

To characterize the fungal isolates, the ITS1-5.8S-ITS2 region of the ribosomal DNA was amplified using the primers described by White et al. (1990), ITS1 5′-TCCGTAGGTGAACCTGCGG and ITS4 5′-TCCTCCGCTTATTGATATGC. The PCR was carried out in 20 μl of the final volume containing 1X buffer, magnesium chloride 2.5 mM, dNTPs 200 µM, 10ρM of each primer and 0.5 U Taq polymerase, using a cycle composed of an initial denaturation for 4 min at 94ºC and thirty-five cycles of 40 s at 94ºC, 40 s at 52ºC, and 40 s at 72ºC, with a final extension of 10 min at 72ºC.

The PCR products were checked on agarose gels at 2%, sequenced using the Macrogen Korea service and analyzed with the MEGA6 program. The sequences were contrasted against the database of the NCBI (http://www.ncbi.nlm.nih.gov/) using the BLASTn tool. The sequences were checked and corrected before being deposited in the GenBank database

All sequences were analyzed using BioEdit and Clustal W before tree construction. A phylogenetic analysis was carried out using the program TNT (tree analysis using New Technology), which performs phylogenetic analysis of parsimony (Goloboff et al., 1999). The gaps (indels) were treated as a fifth state since they represented insertion-deletion events. The analysis included twenty-four sequences of the genus Ascosphaera spp. Aspergillus flavus (NG_055742) was used as an outgroup. The data set was reduced in the heuristic search by implementing 100RAS and guarding a tree by TBR. To evaluate the support in identifying groups, we conducted bootstrap and jackknife parsimony analyses. Both analyses included 1000 resampled matrices. For each resampled matrix, we performed one hundred RAS-TBR cycles (Fonseca et al., 2016).

Argentinian samples of corbicular pollen were disintegrated and homogenized with a mortar and 0.5 g of the total was taken for additional treatment with acetolysis (Erdtman, 1960). The pellet was glycerin mounted and paraffin sealed. Observations were made with a Boeco optical microscope. To determine frequency classes, 600 pollen grains were counted (Louveaux et al., 1978). Pollen types were compared and identified to the palynological deposits from the palynology laboratory of the Faculty of Agrarian Sciences, National University of Jujuy, and by consulting the palynological atlas.

The bees used to carry out the assays belong to the hybrid subspecies A. m. scutellata, present in the province of Jujuy, Argentina, as reported by Porrini et al. (2020) in this region. The bees used for the assay were maintained in Langstroth hives on an experimental farm in Severino (Faculty of Agrarian Sciences of the National University of Jujuy), located in El Carmen department, Jujuy province.

We used three blind cores (no initiating queen), that had been prepared in the spring of 2016 and contained a standard open breeding frame, a breeding frame operculated with nurse bees and a frame with honey, as proposed by Ahmed (2008). We waited until the birth and posture of the queen. When the nuclei grew and consolidated, with about 10,000 bees per frame, they were transferred to three standard brood boxes and allowed to grow again until we obtained a population of approximately 50,000 bees, as proposed by Audisio & Benítez-Ahrendts (2011). Above this brood nest, we placed a medium super frame (without a queen excluder, to allow the queen to pass), which contained twenty-four experimental frames specially designed for assays, measuring 20×10 cm. When the worker bees stretched the beeswax, the posture of the queen was observed, and then when the first bees were born, the state of the hives was monitored monthly. Trials were conducted in the late spring of 2017 and 2018 when temperatures were below 20ºC, the morning temperature registered in this season in Jujuy province. The three hives used for the study were eliminated at the end of the trial to prevent the spread of the disease.

Portions of each fungal strain inoculated with agar and mycelium (about 36 mm²) were placed on Petri dishes in the middle of the MY20 culture and the fungi were grown for ten days until the maturation of sporocysts, under microaerophilia conditions at 30±1ºC. Microscopic observation was performed to confirm the presence of mature spores with a 40 X objective.

An aliquot was taken from each A. apis strain and suspensions were made in tubes with 5 mL of sterile water-peptone (10 g of peptone, 5 g of sodium chloride in 1 liter of distilled water). To release the asci and ascospores contained in the sporocysts, each tube containing glass beads was vortexed for thirty seconds. Counts were performed using a Neubauer chamber, with the concentration adjusted between 5×10³ spores/mL (minimum) and 5×10⁶ spores/mL (maximum). These suspensions were kept at 5ºC until their administration (Jensen et al., 2013; Ansari et al., 2017).

Before being administered to the hives, the ascospore suspensions were spun at 4000 rpm for 5 min and the cell pellets were resuspended in 5 mL of sterile water and honey (1:1). For the control groups (without ascospores), a suspension of 5 mL of sterile water and honey was used (1:1).

The brood chambers of the three assayed colonies were checked before the administration of the ascospores and showed no symptoms or signs of the disease. The uniform posture of the queen and the presence of larvae were verified. Ten open breeding trial frames containing 5- to 6-day-old larvae were selected for the inoculation of the fungal strains. Two repetitions per area of 20×10 cm were performed and areas with capped and uncapped cells were counted before the administration of the strains.

Subsequently, all larvae were fed using a sterile 2.5 mL syringe. They were divided into three groups; the first two were administered a drop of approximately 5 μL of each ascospore suspension at different concentrations: at the minimum concentration and the maximum concentration, respectively, while the third group was used as control (food without ascospores) (See Tab. 3). After feeding, the frames were taken to the original hive so that the cells could be covered by worker bees. At 48 h, the frames were removed from the apiary and incubated in the laboratory for ten days in hives designed for the frames. The mummified larvae in each cell were then counted. To replicate the humidity and temperature environmental conditions in the laboratory, we used a stove at 30±1ºC with 60% humidity.

Data on the number of infected larvae were expressed as mean ± standard deviation of each assay. Statistical analysis of results was performed using ANOVA and Tukey’s mean comparison test, with a 0.5 probability of making a type I error, using Infostat as the statistical package (Di Rienzo et al., 2008).

Three strains of the genus A. apis were isolated from pollen grains of the provinces of Jujuy, Misiones, and Entre Rios, Argentina. They were identified by their macro and microscopic characteristics (Tab. 1, Fig. 2). The development of the sporocysts was observed at five days of incubation. The mycelium exhibited a white cottony, aerial, mostly bright consistency, and its hyphae were covered with droplets of “exudate”. When the asci ripened, they looked black. We observed microscopic spherical brown sporocysts, containing globose asci whose ascospores were hyaline and ellipsoid. The provinces of Tucumán, Buenos Aires, San Luis, Corrientes and Santa Fe had not registered the presence of A. apis in commercial pollen, but strains belonging to the genera Aspergillus, Fusarium, Penicillium, Rhizopus and Mucor were isolated and identified.

Fig. 2

The A. apis strains corresponding to the three provinces yielded polymorphic fragments of 304bp for the Misiones strain, 482bp for Entre Ríos, and 610bp for Jujuy, and shared 100%, 98.3%, and 99.02% identity, respectively, with other strains from the NCBI database. They were registered with the following access numbers: MH633694 (Misiones), MH633695 (Entre Ríos), and MH633693 (Jujuy).

In addition, when the botanical origin of each pollen sample was identified, interestingly, the predominant pollen belonged to the genus Rapistrum and the family Amaranthaceae. While the species Helianthus annus was only recorded in the pollen of Entre Ríos and Misiones, a large percentage of pollen from Jujuy corresponded to the family Myrtaceae (Tab. 2, Fig. 3).

Fig. 3

A total of 872bp were aligned, 299 sites were considered monomorphic, 504 sites were considered polymorphic or phylogenetically informative segregants, and 359 polymorphic sites had more than two variants. The parsimony analysis resulted in two parsimonious trees, with 1889 steps.

Ascosphaera spp. strains were grouped into different phylogenetically related groups. Thus, all A. atra strains shared a common ancestor with 100% support in the bootstrap and jackknife processes. The A. atra and A. duoformis strains also shared a common ancestor with the species A. subcuticulata, A. solina, and A. osmophila with 75% jackknife support and 70% bootstrap support (Fig. 4).

Fig. 4

The A. apis strain and other species of the Ascosphaera genus have a common ancestor with 100% jackknife support, while for the bootstrap process it is located next to the strains of Russia, Australia, the United States and all other Spanish strains. This group contains the strains from Entre Ríos and Misiones. By contrast, the strain from Jujuy belongs to another monophyletic group with strains from the Netherlands and share an immediate common ancestor with those of the United States and the Spanish strains A. apis M05 and M10. Furthermore, the A. celerrima and A. flava strains formed a monophyletic group with similar supports separating from the species of A. apis and A. atra (Fig. 4).

The minimum concentration of ascospores administered in sugar syrup did not differ significantly (ANOVA, p=0.83) in the number of mummified larvae. No mummified larvae were detected in connection with the Entre Rios or Misiones strains, and only the Jujuy strain showed the presence of 1±1 mummified larva (Tab. 3). In contrast, when the suspension with the maximum concentration of ascospores was administered to the larvae, the registered number of mummified larvae with each of the fungal strains was higher (Misiones 30±7, Entre Rios 10±1 and Jujuy 23±3) than the number of mummified larvae fed the minimum concentration of ascospores (Tab. 3). The number of mummified larvae with high concentrations of ascospores exhibited significant differences between the pathogenic effects of the strains from each province (ANOVA, p<0.05) (Fig. 5). The A. apis strain from Entre Rios affected the lowest number of mummified larvae, with percentages close to 11%, while those from Jujuy and Misiones registered infection values close to 27% and 23%, respectively (Tab. 3). The strains from Misiones and Entre Ríos produced 2% of mummified larvae without sporocysts (white mummies), while the strain from Jujuy caused 6%. Closed cells previously capped by bees were not significant for the trial.

Fig. 5

The larvae inoculated with A. apis strains were mummified between days 7 and 10 of incubation. The size of the mummified larvae of worker bees was between 7.5 mm and 8 mm, while that of drones was between 11 and 13 mm (Fig. 2).