Effect of Avid ® on the synnema-like formation of Aspergillus flavus grown on Czapek medium

1 Biotechnology Program, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand. 2 Department of Botany, Faculty of Science, Plant Biomass Utilization Research Unit, Chulalongkorn University, Bangkok, 10330, Thailand. 3 Department of Plant Pathology and Crop Physiology, LSU Agcenter, Louisiana State University, Baton Rouge, Louisiana 70803, USA. 4 Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey 08901-8525, USA.


INTRODUCTION
Aspergillus flavus is a fungus in Aspergillus subgenus Circumdati section Flavi.This species can be divided into two groups based on their aflatoxin production, the aflatoxigenic group (with the ability to produce aflatoxins) and non-aflatoxigenic group (without ability to produce aflatoxins) (Chang and Hua, 2007).Aflatoxigenic A. flavus strains are able to produce aflatoxins B (AFB1 and AFB2) and some strains also produce aflatoxins G (AFG1 and AFG2) (Horn and Greene, 1995).This species is of interest since it can be found in several economically important crops including corn, cotton, peanut and tree nuts, in the field or during storage and is more frequent than other aflatoxigenic strains, such as A. parasiticus, A. nomius and A. pseudotamaraii (Rodriguez and Mahoney, 1994).
Avid ® is a miticide or insecticide used to control mites or insects in a wide range of important crops (Van Der Geest et al., 2000).It is composed of the active ingredient, abamectin, and three inert solvents, mineral oil, butylated hydroxytoluene (BHT) and n-methylpyrrolidone (Syngenta Crop Protection, Inc., Greensboro, NC, USA).Avid ® has been added in microbial culture media to prevent contamination caused by mites or insects associated with crop samples, and thus proved a useful practice for isolation of microorganisms from crop samples.
In our primary observation, when Aspergillus spp.were cultivated on Czapek agar plates containing Avid ® , it was observed that A. flavus strains, but not other species, could form synnema-like structures.Therefore, in this research, the effect of Avid ® on the induction of the synnema-like structure were evaluated in 61 strains of A. flavus growing on various types of solid culture media.Other related species of Aspergillus within the section Flavi (A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus psuedotamarii, Aspergillus tamarii and Aspergillus oryzae) were included for comparison.

Media and culture conditions
Each Aspergillus species / strain was grown on V8 medium (5.0% (v/v) V8 juice and 2.0% (w/v) agar) in the dark at 30°C for 7 days.Spores were harvested from these cultures using sterile water and the suspension was adjusted to 10 5 spores per ml.A spore suspension of each Aspergillus species (5 µl per plate) was inoculated onto solid media and incubated in the dark at 30°C for 7 days for morphological observation.
To investigate the effect of Avid ® (0.15 EC, Norvatis Crop Protection, Greensboro, NC, USA) on the synnema-like formation of A. flavus, and the other related species in the section Flavi, each fungal isolate was grown on Czapek, V8, AFPA (Aspergillus flavus and parasiticus agar) (Pitt et al., 1983), Corn Meal Agar (CMA, Difco, USA) and Potato dextrose Agar (PDA, Difco, USA), with or without 0.004% (v/v) Avid ® , incubated in the dark at 30°C for 7 days.To determine the effect of the Avid ® concentration on the synnema-like formation, Avid ® at six different concentrations (0.002, 0.004, 0.008, 0.02, 0.05 and 0.01 % (v/v)) was added into the culture medium.Carbon and nitrogen sources in the culture Danmek et al. 2813 medium were varied so as to include one of each three carbon sources (sucrose, glucose and starch) and three nitrogen sources (NaNO3, urea and peptone).The three inert solvents in Avid ® , mineral oil, butylated hydroxytoluene (BHT) and nmethylpyrrolidone (0.004% v/v), were also tested for their ability to induce a synnema-like structure by incorporating them into the Czapek medium.The colonies of fungi were observed daily for morphological changes.All experiments were performed in triplicate.

RESULTS AND DISCUSSION
When Aspergillus spp.were grown on different kinds of solid culture media (Czapek, V8, AFPA, CMA and PDA) with or without Avid ® at 0.004% (v/v), all cultures grew well and produced conidia regularly.However, only A. flavus (of all 61 strains) grown on Czapek medium containing Avid ® , developed the synnema-like structures (fused white conidiophores bearing yellow to green conidia) after 3 days of incubation at 30°C in the dark (Figure 1).When this species was grown on Czapek media without Avid ® or on the four other media tested with or without Avid ® , the fungus did not produce a synnema-like structure (Figure 2).In this study the miticide clearly did not affect the growth of Aspergillus spp.However, Avid ® might be able to decrease the germination, vegetative growth, and conidial number of the entomopathogenic fungus, Beauveria bassiana, (Oliveira and Neves, 2004).Although not commonly found, the formation of synnema by Aspergillus section Flavi was previously reported in a mutant of A. flavus (NRRL 29254) and some strains of A. caelatus which was induced by the regulation of carbon and nitrogen sources in the culture media (McAlpin, 2001;2004).
All strains of A. flavus could grow and form synnemalike structures at different level concentrations of Avid ® , ranging from 0.002 to 0.01% (v/v), added into Czapek media.Thus, Avid ® at this concentration range, can induce the formation of a synnema-like structure of A. flavus.The formation of synnema-like structure of A. flavus was not affected by the three carbon sources tested, with all strains of A. flavus showing no difference in colony morphologies when they grew on Czapek media containing Avid ® using either sucrose, glucose, or starch as the sole carbon source, and they all produced synnema-like structures.However, with respect to the nitrogen source, all A. flavus strains could form synnemalike structures only when grown on Czapek media containing Avid ® with NaNO 3 as the sole nitrogen source.Urea and peptone were not able to induce synnema-like structure in A. flavus.The results support the absence of synnema-like structure of A. flavus strains grown on V8, AFPA, CMA, and PDA media since they contain either urea or peptone as a nitrogen source.The role of nitrogen source in morphological changes of Aspergillus has previously been reported.NaNO 3 was shown to be an excellent nitrogen source for synnema/sclerotium formation while ammonium sulfate inhibited growth, sporulation and synnema/sclerotium formation of A. caelatus and a mutant strain of A. flavus (McAlpin, 2001;2004).
In order to clarify which ingredient in Avid ® could induce the synnema-like structure of A. flavus, the inert solvents in Avid ® , mineral oil, butylated hydroxytoluene (BHT) and n-methylpyrrolidone, were separately added in Czapek medium to the same concentration as that of 0.004% Avid ® .All A. flavus strains failed to produce synnema-like structures when grown on Czapek medium containing any of these three inert solvents.Thus, unless a corequirement of two or more of the inert solvents is required; the result suggests that synnema-like structure of this species was induced by abamectin, an active ingredient in Avid ® .
In conclusion, the results presented here show that Avid ® could induce synnema-like structure of A. flavus growing on Czapek medium.None of the other Aspergillus species tested could form this structure when grown on this medium containing Avid ® .The miticide could induce synnema-like structure at a wide concentration range (from 0.002 to 0.01% (v/v)).NaNO 3 in Czapek medium together with abamectin in Avid ® were found to play important roles in the induction of synnemalike structure of A. flavus.The results open up the possibility of using Avid ® in a selective culture medium to help distinguish A. flavus from the other Aspergillus in the section Flavi.