Beauveria bassiana is a well-known broad-range arthropod pathogen which has been used in biological control of several pest insects and ticks such as Boophilus microplus. Beauveria amorpha has both endophytic and entomopathogenic characteristics, but its capacity for biological control has still not been studied. During the processes of host infection, B. bassiana and B. amorpha produce several hydrolytic extracellular enzymes, including proteases and chitinases, which probably degrade the host cuticle and are suggested to be pathogenicity determinants. To access the role of these enzymes during infection in the tick B. microplus, we analyzed their secretion during fungus growth in single and combined carbon sources, compared to complex substrates such as chitin and B. microplus cuticle. Chitin and tick cuticle-induced chitinase in both fungus and protease was induced only by tick cuticle. SEM analysis of B. amorpha and B. bassiana infecting B. microplus showed apressorium formation during penetration on cattle tick cuticle.

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.

The current study compared the virulence of conidia and blastospores of Metarhizium robertsii (IP 146), M. anisopliae sensu lato (s.l.) (IP 363) and Beauveria bassiana s.l. (IP 361 and CG 307) against unfed larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). In addition, the development of fungal propagules on tick cuticle was investigated. Tick larvae were treated with fungal suspensions at 10, 10 or 10 propagules mL, and percent mortality was assessed every two days. Engorged females were immersed in fungal suspensions (1.0 × 10 propagules mL) for 1 min, and their biological parameters monitored daily. The virulence of conidia and blastospores against larvae varied considerably among the isolates tested. Only females treated with blastospores of IP 146 or IP 361 presented lower nutrient and egg production indices than the control group; the higher percent control was reached when females were treated with blastospores of IP 146 (98%), IP 363 (79%), or IP 361 (93%) in comparison to the groups treated with conidia, 71%, 59%, or 63%, respectively. Engorged females treated with conidia or blastospores of IP 146 or IP 361 were also examined by scanning electron microscopy (SEM). Germination of blastospores of IP 146 and IP 361 was observed on tick cuticle after 4 h incubation at 27 ± 1 °C and RH > 90%, whereas germ tubes from conidia of both isolates were observed at 48 h. Appressoria in developing blastospores of B. bassiana IP 361 were observed after 4 h incubation, whereas no appressoria were seen in developing blastospores of M. robertsii IP 146. Blastospore penetrations by both fungal isolates through natural openings was also evidenced by SEM; fine sections of R. microplus engorged females treated with blastospores of IP 146 or IP 361 confirmed that these isolates penetrated through their cuticle and natural openings. Blastospores might be promising for use in biocontrol of ticks, since they are virulent against R. microplus and present rapid development on their cuticle.

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Crude ethanol extracted from Magonia pubescens trunks was utilized to prove its effect in the intestinal tract of Aedes aegypti 3rd instar larvae. Exposure times (2, 4, 6, 8, 10, 12, and 13 hours) were tested to verify when the morphological alterations begin to occur, in the intestinal tract of the larvae. The toxic effect of the extract was mainly in the midgut, beginning at the anterior midgut and followed through to posterior midgut. The main alterations observed were partial or total cell destruction, high citoplasmatic vacuolization, increase of subperitrophic space, cell hypertrophy and the epithelium did not maintain its monolayer appearance. The alterations began after four hours of exposure to M. pubescens extract. Observations of tissue sections from larvae treated for different lengths of time revealed a wide variation in the degree of damage between exposure periods and midgut larvae region. The present study provides evidence regarding the mode of action of the M. pubescens extract and suggests its potential utilization as a larvicide to control Aedes aegypti mosquito.

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.

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