Ingrid Ney Kramer de Mello

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

Abstract The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 106 conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K2HPO4 (5.0 g/l), MgSO4 (0.10 g/l), ZnSO4 (0.0050 g/l), FeSO4 (0.001 g/l) e CuSO4 (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.

Biological control of infective larvae of Ancylostoma spp. in beach sand.

BACKGROUND Geohelminths are parasites that stand out for their prevalence and wide distribution, depending on the soil for their transmission. AIMS The aim of this work was to evaluate the predatory capacity of the fungal isolate of the genus Duddingtonia (CG768) on third stage larvae (L3) of Ancylostoma spp. in beach sand under laboratory conditions. METHODS In the assay A five treatment groups and 1 control group were formed. The treatment groups contained 5000, 10,000, 15,000, 20,000 or 25,000 chlamydospores of the fungal isolate and 1000 Ancylostoma spp. L3 in pots containing 30g of sand. The control group (without fungus) contained only 1000 Ancylostoma spp. L3 and distilled water in pots with 30g of sand. RESULTS Evidence of predatory activity was observed at the end of 15 days, where we observed the following percentages of reduction of L3: Group 1 (4.5%); Group 2 (24.5%); Group 3 (59.2%); Group 4 (58.8%); Group 5 (63%). However, difference was noted (p<0.01) only at concentrations 15,000, 20,000 and 25,000 in relation to control group. In the assay B two groups were formed in Petri dishes of 9cm in diameter containing agar water 2% medium. In the treated group, each Petri dish contained 500 Ancylostoma spp. L3 and 5g of sand containing the isolate CG 768 at a concentration of 25,000 chlamydospores/g of sand, and the control group (without fungus) contained only 500 L3. At the end of 7 days the non-predation L3 of Petri dishes using the method of Baermann were recovered. Difference (p<0.01) between groups on reducing the average number of Ancylostoma spp. L3 (percent reduction of 84%) was observed. CONCLUSIONS The results of this study confirm earlier work on the efficiency of the Duddingtonia genus in the control of Ancylostoma spp. infective larvae.

First report of the activity of predatory fungi on Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) first-stage larvae☆

The nematode Angiostrongylus cantonensis causes eosinophilic meningoencephalitis in humans and thus alternative methods of control should be studied. The objective of this work was to evaluate the predatory capacity of eight fungal isolates of the species Duddingtonia flagrans (AC001, CG768 and CG722), Monacrosporium thaumasium (NF34), M. sinense (SF53) and Arthrobotrys robusta (I31), A. cladodes (CG719) and A. conoides (I40) on first-stage larvae (L₁) of A. cantonensis under laboratory conditions. The treated groups contained 1000 conidia of the fungal isolates and 1000 A. cantonensis L₁ in Petri dishes containing 2% water-agar medium (2% WA). The control group (without fungi) contained only 1000 A. cantonensis L₁ in 2% WA. Evidence of predation was observed at the end of 7 days. Percentage reductions in L₁ were: AC001, 82.8%; CG768, 71.0%; CG722, 72.8%; NF34, 86.7%; SF53, 89.7%; I40, 48.3%; CG719, 84.7%; and I31, 80.4%. No significant difference was observed (p>0.01) between the actions of the isolates used; however, a difference was noted (p<0.01) in relation to the control group. The results of the present work, confirm previous reports of the effectiveness of the fungi D. flagrans, M. thaumasium, M. sinense and A. robusta in controlling larvae of potentially zoonotic nematodes, this being the first report on A. cantonensis L₁.

Assessment of compatibility between the nematophagous fungi Arthrobotrys robusta and Duddingtonia flagrans under laboratory conditions

Nematophagous fungi are used in the biological control of pests nd diseases.1,2,5,7,10 The use of more than a single biocontrol agent s considered one of the main suppressive measures contributing to ontrol the presence of infectious agents in soils.8 The use of more han one nematophagous fungi in combination can minimize posible flaws in their administration or even enhance their actions as iocontrol agents.6 However, biological control mechanisms such s the capability to produce substances having a fungicidal effect, ay vary among species and even among isolates of the same pecies during the life of the antagonist.9 This work aimed to study the compatibility between rthrobotrys robusta and Duddingtonia flagrans under laboraory conditions by means of direct confrontation, antibiosis and olatile metabolites tests. Two predatory fungi, D. flagrans (AC001) and A. robusta (I31) ere used. A culture disc containing AC001 and a disc with 31 were placed on opposing sides of a Petri plate in culture edium containing 2% potato dextrose agar. These plates were ncubated for 10 days at 26 ◦C in the dark and, after this period, ere evaluated with a rating scale.3 The experiment had five epetitions. The antibiosis test followed the modified methodology of Martins-Corder and Melo.9 The plates were then kept at 26 ◦C in the dark. After 10 days of incubation, we assessed the formation of inhibition zones.9 The effect of volatile metabolites was evaluated with Bharat et al.4 methodology. The plates were sealed laterally and maintained at 25 ◦C for five days in the dark. The area of each colony was measured and then the data were submitted to ANOVA and Tukey’s test at significance levels of 1% and 5%, and the degrees of freedom of the treatments deployed in orthogonal contrasts. The experiment, in a completely randomized design, had four replications. Two aliquots of the same species of fungus grown in the same plate showed homogeneous growth in a direct confrontation test, confirming the non-interference of one colony on the other (Fig. 1A and C). However, in the test with A. robusta and D. flagrans, I31 colonized approximately 2/3 of the plate, suggesting competition between them and therefore some antagonism (Fig. 1B). However, there was no evidence of hyper parasitism without growth of a colony on each other. The production of toxic metabolites by D. flagrans and A. robusta is unknown; therefore, it was important to show that they do not produce compounds capable of inhibiting one another in a joint application. This was verified by an absence of antibiotic production by both fungi in the culture medium, as a result of which there were no zones of inhibition when a culture was subsequently sown on the same plate. Nevertheless, A. robusta reduced the growth of D. flagrans, suggesting the action of volatile antibiotics in inhibiting mycelial growth. Furthermore, D. flagrans did not reduce the growth of A. robusta (Table 1).

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L3 in the media supplemented with casein and pH 7.0.

Biological control on gastrointestinal nematodes in cattle with association of nematophagous fungi

ABSTRACT This study aimed to evaluate the efficacy of the association of the nematophagous fungi (Duddingtonia flagrans-AC001); (Pochonia chlamydosporia-VC4) and (Arthrobotrys robusta-I31) in a pelletised formulation of a sodium alginate matrix. The viability and activity of pellet germination and fungal activity (after encapsulation) were evaluated using in vitro and in vivo tests. Next, 12 heads of cattle, Dutch mestizo x zebu, with an average age of 12 months were dewormed with an anthelmintic. Next, 20 days after treatment with the anthelmintic, the animals were randomly divided into two groups of six animals each, and placed in two paddocks with 7.0 ha each of Brachiaria decumbens with historical grazing by animals naturally infected by gastrointestinal nematode parasites. At first, each animal was treated with 2 g of pellets per 10 kg of animal, containing the associated fungi (AC001 + VC4 + I31) administered twice a week in conjunction with commercial feed. Each animal in the control group received 2 g of pellets without mycelia added to the feed. The percentage reduction of infective larvae in the in vitro test was 94% (p < .01). In the in vivo test, the treated animals with fungal association had lower egg counts per gram of faeces (p < .01) compared to the control group animals – a reduction of 91.8%. The reduction in the number of infective larvae recovered from the pasture was 27.5% at a distance of 0–20 cm from the stool bulk, and 26.7% at a distance of 20–40 cm. This association (AC001 + VC4 + I31) of nematophagous fungi was effective in nematode control.