Toxin Reviews, 2009; 28(2–3): 142–153

REVIEW ARTICLE

Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies to reduce a atoxin contamination of corn

HK Abbas1, JR Wilkinson2, RM Zablotowicz3, C Accinelli4, CA Abel5, HA Bruns1, and MA Weaver3

1USDA-ARS, Crop Genetics and Production Research Unit, Stoneville, MS, 2Mississippi State University, Department of Biochemistry and Molecular Biology, MS, 3USDA-ARS, Southern Weed Science Research Unit, Stoneville, MS, 4University of Bologna, Department of Agro-Environmental Science and Technology, Bologna, Italy, and 5USDA-ARS, Southern Insect Management Research Unit, Stoneville, MS

Abstract The contamination of corn (maize) by fungi and the accumulation of mycotoxins are a serious agricultural problem for human and animal health. One particular devastating group of mycotoxins, called a atoxins, has been intensely studied since the 1960s. Studies of Aspergillus avus, the agriculturally relevant pro- ducer of a atoxins, have led to a well-characterized biosynthetic pathway for a atoxin production, as well as a basic understanding of the organism’s life cycle. Unfortunately, these eorts have not resulted in corn production practices that substantially reduce a atoxin contamination. Similarly, the use of agrochemicals (e.g., fungicides) results in very limited reduction of the fungus or the toxin. Thus, cultural management (fertility and irrigation) coupled with aggressive insect management is current recommendation for inte- grated a atoxin management. The development of resistant hybrids appears to be a very promising tech- nology, but commercial hybrids are still not available. Thus, biocontrol appears to be the most promising available avenue of reducing a atoxin accumulation. Biocontrol utilizes nontoxigenic strains of Aspergillus to reduce the incidence of toxin-producing isolates through competitive displacement. To maximize the eectiveness of biocontrol, a thorough knowledge of the environmental factors in uencing colonization and growth of Aspergillus is needed. A. avus not only colonizes living plant tissue, but it also grows sapro- phytically on plant tissue in the soil. These residues serve as a reservoir for the fungus, allowing it to over- winter, and under favorable conditions it will resume growth and release new conidia. The conidia can be transmitted by air or insects to serve as new inoculum on host plants or debris in the eld. This complex ecology of Aspergilli has been studied, but our understanding lags behind what is known about biosynthe- sis of the toxin itself. Our limited understanding of Aspergilli soil ecology is in part due to limitations in eval- uating Aspergilli, a atoxin, and the biosynthetic genes in the varying aspects of the environment. Current methods for assessing Aspergillus and a atoxin accumulation rely heavily on cultural and analytical meth- ods that are low throughput and technically challenging. Thus to understand Aspergillus ecology and envi- ronmental eects in contamination to maximize biocontrol eorts, it is necessary to understand current treatment eects and to develop methodologies capable of assessing the fungal populations present. In this manuscript we discuss the current knowledge of A. avus ecology, the application of selected molecu- lar techniques to eld assessments, and crop practices used to reduce a atoxin contamination, focusing on chemical treatments (fungicides and herbicides), insect management, and crop management. Keywords: Fungal ecology; a atoxin; insect; corn; mycotoxin; biological control; environmental manipulation; molecular biology; fungal biology

Introduction a atoxins and other chemicals with deleterious properties (e.g., aspergillic acid, cyclopiazonic acid, Aspergillus avus is a fungus of economic and kojic acid, helvulic acid, etc.). is fungus is ubiq- toxicological importance due to the production of uitous in the environment, being readily isolated

Address for Correspondence: Dr. HK Abbas, USDA-ARS, Crop Genetics and Production Research Unit, Stoneville, MS 38776, USA. E-mail: [email protected] (Received 06 March 2009; revised 03 April 2009; accepted 06 April 2009)

ISSN 1556-9543 print/ISSN 1556-9551 online © 2009 Informa UK Ltd DOI: 10.1080/15569540903081590 http://www.informapharmascience.com/txr Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 143 from plants, air, soil, and insects (Wicklow et al., Contamination of agricultural commodities by 2003). e presence of Aspergillus species (A. avus, a atoxin is a serious problem due to the substantial A. fumigatus, and A. vesicolor) in dust can compro- health eect it has on humans and animals. Several mise individuals with elevated allergies to the fungus strategies that have been employed to minimize or its products (Benndorf et al., 2008; Sharnma et al., a atoxin contamination are listed in Table 1. e 2007). Of more concern is the colonization of certain use of agrochemicals (fungicides), timely irrigation, food or feed crops (corn, cottonseed, peanuts, and and alternate cropping systems have independently some tree nuts) by the fungus, where it may pro- shown limited success in preventing a atoxin con- duce a high enough concentration of these chemi- tamination. Integration of several of these tactics cal products, specically a atoxin, to cause them will be required to manage such a dicult problem to be considered contaminated and unt for their (Cleveland et al., 2003). e use of these management intended use. techniques will be further discussed in the following A atoxin was initially identied as toxic after sections of this article. A more recent and promis- investigations of the death of 100,000 turkeys in the ing technology is the use of nontoxigenic strains of United Kingdom in 1960 (Blout, 1961). is prompted Aspergillus as biocontrol agents. However, to maxi- a major revolution in mycotoxin research result- mize this methodology and to prevent the coloniza- ing in intensive testing for mycotoxins in any moldy tion of multiple crops by A. avus and related spe- products. Since then several Aspergilli have been cies (A. parasiticus and A. nominus), it is critical that identied as capable of producing a atoxins. e two a complete understanding of the ecology of these most agriculturally important species are Aspergillus unique fungi be developed. avus and A. parasiticus, which are found through- rough the use of sophisticated molecular biol- out the world, being present in both the soil and the ogy approaches, our knowledge of Aspergillus biol- air (Abbas et al., 2005; Klich, 2002; Horn and Dorner, ogy and a atoxin management continues to improve. 1998; Wicklow et al., 1998). When conidia (spores) As we understand the genes and gene expression encounter a suitable nutrient source and favorable of these fungi and their interactions with the plant environmental conditions (hot and dry conditions) host, we will improve control mechanisms on several the fungus rapidly colonizes and produces a atoxin fronts (Table 2). us, new solutions to reduce a a- (Payne, 1992). toxin contamination will result through the coupling

Table 1. Crop, insect, and soil management practices to manage a atoxin contamination. Strategy Method Rationale Avoidance Early planting, supplemental irrigation, short season Reduce heat and moisture stress hybrids Fertility management Provide adequate nutrition N-decient corn more susceptible Insecticide application Appropriate timing of application to control insect damage to Insects responsible for enhanced ingress into ears grain Bt Hybrids Hybrids engineered with resistance to ear-damaging insects Insects responsible for penetration into grain Natural resistance Breeding and selection hybrids for resistance to insects Biological control Use of nontoxigenic isolates of A. avus Competitive displacement of toxigenic isolates Fungicides Control phylosphere fungi Reduce inoculum density Soil management Incorporation of crop residues Reduce inoculum density

Table 2. Application of molecular methods in a atoxin research. Goal Methods Rationale Identication of a atoxin EST sequence, whole genome sequence, cDNA Reduce a atoxin production by manipulating biosynthetic and regulatory genes microarrays, whole genome microarrays factors that induce a atoxin biosynthesis Identication of Aspergillus A. avus microarrays, Z. mays microarrays, Develop markers for selection of hybrids with pathogenicity factors and maize qRT-PCR, digital gene expression, resistance to Aspergillus infection and a atoxin resistances factors to Aspergillus accumuation Identication of maize resistances Z. mays microarrays, qRT-PCR, digital gene Develop markers for selection of hybrids with factors to Fall Army Worm expression resistance to ear-damaging insects to reduce Aspergillus vectoring Develop molecular identication SSR ngerprinting, PCR identication of rRNA, Track soil populations during biocontrol to methods RT-PCR of biosynthetic genes improve displacement of toxigenic strains 144 HK Abbas et al. of traditional agronomic research with these modern Cary and Calvo, 2008), and no complete regulatory tools. model has been validated.

A atoxin and biosynthesis Biology and ecology of Aspergillus avus

A atoxins are toxic compounds chemically related Generally, Aspergillus avus is a competent sapro- to bisfuranocoumarin that are produced by A. avus phyte and can readily survive and colonize soil and and A. parasiticus strains (Abbas et al., 2004b; Payne, organic debris associated with plant residues (Abbas 1992). ese two a atoxigenic species have been fre- et al., 2008a; Jamie-Garcia and Cotty, 2004). e over- quently studied due to their impact on agricultural wintering reservoir of A. avus propagules in soils commodities and their devastating eects on live- and plant debris can serve as a primary inoculum for stock. e name “a atoxin” comes from the genus infestation of below-ground plant parts, especially Aspergillus, which is where the letter “a” in a atoxin in peanuts. Although soil can serve as the primary is derived and “ a” from the species name avus. In habitat for A. avus, A. parasiticus, and A. nominus, agricultural grains the fungi A. avus and A. para- little is known about the life cycle of these fungi in siticus are capable of producing four major a atoxins soil. A. avus is capable of surviving and overwinter- (AfB1, AfB2, AfG1, and AfG2). A. avus typically pro- ing in plant residues as mycelium (hypha) or sclerotia duces only the B toxins (Abbas et al., 2004b; Fortnum, (Abbas et al., 2008a; Horn, 2007; Payne, 1998; Wicklow 1986; Heathcote and Hibbert, 1978; Hill et al., 1985; et al., 1993) that in turn serve as the source of new Jones et al., 1981; Manwiller and Fortnum, 1979; conidia (Figure 1, life cycle of A. avus). Payne, 1998). Corn and cottonseed are typically con- Soil populations of A. avus in soils under maize taminated with the a atoxin B1, produced after colo- cultivation can range from 200 to >300,000 colony- nization by A. avus (Hill et al., 1985; Klich, 1986). forming units (cfu) g−1 soil (Abbas et al., 2004a, A. parasiticus is more prevalent in peanuts than any Zablotowicz et al., 2007) and can constitute from other crop; however, it is typically outcompeted by ≤0.2% to ≤8% of the culturable soil fungi population. A. avus when the two fungi are both present (Horn e major soil property associated with maintaining et al., 1995). soil populations of A. avus is soil organic matter. ese toxins were named based on their physical As shown in Figure 2, a highly signicant correla- characteristics of uorescence under longwave ultra- tion between soil populations and organic matter violet light (L=365nm) when initially isolated using is observed. e four major outlier points consist of thin-layer chromatography; AfB1 and AfB2 produce a one soil that had several-fold higher organic matter blue uorescence, whereas AfG1 and AfG2 produce a content compared with the other soils. is soil was green uorescence (Klich, 2007). ere are close to 25 part of a comparison of intensive crop residue man- analogs of a atoxins; among these the most impor- agement practices. From this study it was also evident tant to animals are a atoxin B1-2,3-oxide (AfB1-2/3- that higher populations of A. avus are maintained in oxide) and a atoxin M1 (AfM1, which is a derivative the soil surface of no-till compared to conventional- of AfB1, formed in dairy cattle milk) (Heathcote and till soils (Zablotowicz et al., 2007). Hibbert, 1978; Bhatnagar et al., 2002, 2006a, 2006b). Following harvesting of a maize crop, A. avus e biosynthetic pathway in A. avus consists propagules range from greater than log 3 to ^log of approximately 23 enzymatic reactions and at 6cfu g−1 tissue (Table 3) (Abbas et al., 2008a). Similar least 15 intermediates (reviewed in Bhatnagar et al., levels are seen in vegetative tissue from soybean and 2003, 2006a; Minto and Townsend, 1997; Payne and cotton residues as observed in corn stover (Abbas, Brown, 1998; Yu, 2004; Yu et al., 2002) encoded by 25 unpublished). However, typically >log 5cfu g−1 tis- identied genes clustered within a 70-kb DNA region sue are associated with maize cobs and maize cobs on chromosome III (Bhatnagar et al., 2006a; Cary and containing grain 3 months after harvest, while levels Ehrlich, 2006; Smith et al., 2007; Townsend, 1997; Yu of ^log 2.5cfu g−1 or lower are observed in soybean et al., 1995, 2004, 2005). e initial substrate acetate is seed (Table 3). Clearly, abundant propagules of used to generate polyketides with the rst stable path- A. avus can be maintained on the soil surface of way intermediate being the anthraquinone norsolo- no-till corn elds. Although it is wise, from a soil rinic acid (NOR) (Bennett, 1981; Bennett et al., 1997). conservation standpoint, to utilize no-till practices to is is followed by anthraquinones, xanthones, and maintain soil resources, such practices may enhance ultimately a atoxins synthesis (Bhatnagar et al., 2003, the potential for a atoxin contamination. e data 2006a; Yabe, 2003; Yu et al., 2002, 2004). Few regula- on A. avus propagule levels in nonhosts such as tors of this process have been identied (reviewed in soybean suggest that maintaining residues of other Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 145

Wind Pathogenic Stage Secondary Inoculum

Insects Wind

Insects

Conidia

Sclerotia

Sporulating mycelia

Plant residue

Soil Saprophytic Stage Mycelia Conidia

Figure 1. Life cycle of A. avus in a corn cropping system saprophytic and pathogenic stages of fungal ecology.

5 Table 3. A. avus propagules, toxigenic isolates, and a atoxin concentration in various residue fractions of Bt and non-Bt corn, r2 = 0.4598 Bt cotton, and soybean residues, collected in December 2006. 4 Pr > F = <0.001 Total Colony-forming Toxigenic a atoxin 3 units (log 10 isolates content Source propagules g−1) (% of total) (Mg kg−1) 2 Bt corn cobs with 5.7±0.3 51±24 542±266 grain Bt corn cobs 5.1±0.4 34±25 17±10

Soil organic matter (%) 1 Bt corn stover 3.7±0.1 64±16 3.3±1.7 Non-Bt corn cobs 5.6±0.3 46±19 774±195 0 with grain 2.0 2.5 3.0 3.5 4.0 4.5 Non-Bt corn cobs 5.5±0.7 33±21 105±96 Aspergillus flavus log (10) cfu g–1 Non-Bt corn stover 3.9±0.3 47±15 3.9±2.5 Cotton Bt 3.5±0.4 73±19 0.7±0.5 Figure 2. Relationship between soil organic matter content and populations associated with Mississippi maize-producing soils Soybean (non-Bt) 3.5±0.4 22±14 0.2±0.4 (Zablotowicz et al., 2007). Soybean grain 1.8±0.3 15±14 0.2±0.4 (non-Bt) crops on the soil surface may be suitable to reduce inoculum potential of A. avus. Before consideration Zablotowicz et al., 2007). When using cultural tech- of alternative cover crop management with crops that niques for enumeration such as serial dilution tech- may be allelopathic to A. avus, further investigation nique and plating, it is dicult to ascertain whether into Aspergillus antagonism is necessary. propagules enumerated are present as hyphal frag- Corn samples mean and standard deviation of ve ments or as spores. Sieving techniques can be used replicates, cotton and soybean mean and standard for the recovery of sclerotia from soil or plant tissue deviation of four replicates. homogenates; however, the actual identication of ere are few methodologies, all of which are species can be dicult as sclerotia of a given species quite tedious, available to ascertain if propagules of can vary tremendously in size and shape (Klich and Aspergillus are actively growing in soil or present in Pitt, 1988; Abbas et al., 2005; Horn, 2003). quiescent propagules, e.g., conidia or sclerotia. e When suitable environmental conditions arise, majority of studies that evaluate A. avus soil ecol- sclerotia and conidia germinate into mycelia that pro- ogy have traditionally used selective plating tech- duce numerous conidiophores and release conidia niques (Abbas et al., 2004a; Horn and Dorner, 1998; into the air that can be available for colonizing plants. 146 HK Abbas et al.

Although A. avus colonizes a plant structure, it near cultivated land are observed to have very low doesn’t necessarily produce a atoxin to excessive populations of A. avus (Angle et al., 1982; Horn, levels. In this manner, A. avus is an opportunistic 2007). Similarly, the frequency of drought is a factor pathogen in a similar context to the opportunistic in populations of fungi, with signicant drops in soil human pathogens Pseudomonas uorescens and populations of A. avus after several years without Burkholderia cepacia. ese bacteria may colonize in drought (Horn and Dorner, 1998; Horn, 2007). e low levels in compromised individuals, such as burn conidia remain dormant in soil and only germinate patients or the immunocompromised, and become when nutrient sources are present. is is supported pathogenic. In the same context, healthy plant tissues by several studies where the A. avus population are less prone to be extensively colonized by A. avus. densities increased with residue managed systems However, under heat stress and moisture decit, corn (Abbas et al., 2004b; Grin et al., 1981; Horn et al., reproductive structures are readily susceptible to high 1995; Zablotowicz et al., 2007). levels of a atoxin contamination (O’Brian et al., 2007). Based on the observation of natural variability in erefore, inoculum potential modied by life cycle of a atoxigenic strains, nona atoxigenic isolates have the fungus is as critical as the environment and host. been used as biological control agents to control a a- e A. avus life cycle can be divided into two toxin contamination (Abbas et al., 2006, 2008c; Cotty, major phases: the colonization of plant residues in 1994; Dorner et al., 1999;). If the introduced nona a- soil, and the infection of crop tissues, including grain toxigenic isolate can adequately colonize and com- and seeds of actively growing plant tissues. At the petitively displace the toxigenic isolates, the level of beginning of the growing season, usually in spring contamination is reduced (Abbas et al., 2008b and c). and sometimes at the end of winter, when sclerotia Although biological control treatments may alter the are exposed to the soil surface, they quickly germinate levels of toxigenic and nontoxigenic isolates, the exact and form new conidial inoculum. is new inoculum nature of these interactions is unknown. A summary will be vectored by insects or carried by the wind to of mechanisms to exploit various biocontrol strate- begin the colonization and infection of the freshly gies in reducing a atoxin contamination is presented planted crops (Horn, 2007; Payne, 1992). During in Table 4. e behavior of Aspergilli structures in soil the growing season, infected plant tissues can serve needs to be investigated and evaluated thoroughly, as sources of secondary conidial inoculum, which especially in agricultural soils, due to fungal structures colonize new noninfected plant tissues (Figure 1, life serving as the primary inoculum resulting in a atoxin cycle). Despite our understanding of how the initial contamination in agricultural commodities. and secondary inocula occur for plant infection, lit- tle information is available about the saprotrophic Table 4. Biological control approaches to reduce a atoxin activities of these fungi in soil. Recently, Accinelli contamination in corn and other crops. et al. (2008c) conrmed the presence of A. avus in Approach Crop Results Citation the soil actively synthesizing a atoxins. Accinelli et al. Use of grain Corn Up to 86% reduction Abbas reported that transcription of 5 a atoxin biosynthesis colonized by in a atoxin et al., 2006 genes, including a D, a G, a P, a R, and a S, were nontoxigenic contamination of detected by reverse transcription-polymerase chain A. avus strains maize reaction (RT-PCR) analyses in soil. However, not all Use of grain Cotton EPA registration, Cotty colonized by non- production and area- et al., 2007 A. avus and A. parasitcus isolates produce a atoxins toxigenic A. avus wide deployment of (Abbas et al., 2004b). strains AF36 Fungi are classied as nona atoxigenic if they do Use of grain Peanuts Competitive exclu- Dorner, not produce a atoxins but produce other toxins. If coated with non- sion of toxigenic 2008; fungi produce no toxins at all, they are classied as toxigenic A. avus strains and reduction Dorner nontoxigenic. Generally, in any environment, the fre- strains of a atoxin in peanut et al., 2003 both pre- and post- quency of a atoxigenic isolates can range from 50% harvest. to 80% (Abbas et al., 2004a, 2004b, 2005; Pildain et al., Use of foliar Corn Greater than 90% Lyn et al., 2004). e relative distribution of a atoxigenic versus application of non- colonization of maize 2009 nona atoxigenic isolates is modulated by many fac- toxigenic kernels by non- tors including plant species present, soil composition, A. avus strains toxigenic strain when applied as conidia cropping history, crop management, and environ- or as a formulated ment conditions, including rain fall and temperature conidial product (Abbas et al., 2004b; Horn et al., 1995; Zablotowicz Yeast Nuts Identication of Campbell et al., 2007). Each of these factors can reduce the antagonists of et al., 2003 levels of A. avus, for example, noncultivated elds a atoxin production Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 147

Molecular biology applications to used to detect the presence of A. avus in agricultural Aspergillus ecology and a atoxin commodities and environmental samples, including contamination soil and insects (Accinelli et al., 2008a, 2008b; Criseo et al., 2001; Geisen, 1996; Manonmani, et al., 2005). e dependence of A. avus on crops to maintain soil Recently completed and ongoing studies using populations emphasizes the importance of under- molecular techniques are beginning to greatly increase standing the relationship between stress physiology our knowledge of A. avus population structures in of the crop and this opportunistic fungus. To achieve soil. PCR has been employed to identify the presence a more complete picture of Aspergillus spp. ecology, of Aspergilli in the soil by Accinelli et al. (2008a), while rapid methods of identication are needed so that RT-PCR (reverse transcription-polymerase chain a broader array of conditions can be investigated. reaction) has also been employed to distinguish toxi- Unfortunately Aspergillus species are not suitable genic and nontoxigenic A. avus (Degola et al., 2006; for molecular techniques such as uorescent in situ Scherm et al., 2005), as well as to track the presence hybridization (FISH). e molecular ecology of sev- of a atoxin biosynthetic genes (Accinelli et al., 2008a eral bacteria and fungi has been advanced by FISH and b). e novel technique of pyrosequencing may coupled with autoradiography; however, the nucle- prove useful in quantifying the presence of introduced otide probes are unable to penetrate through the cell A. avus strains in mixed communities of A. avus walls of Aspergillus (Teertsta et al., 2004). us, the (Das et al., 2008). We anticipate future ecological majority of ecological population studies rely upon tests to utilize and expand these molecular methods enumeration and vegetative compatibility groups to examine Aspergilli populations and biochemical to characterize A. avus distributions (Bayman and behaviors in soil. In conclusion, Aspergillus soil ecol- Cotty, 1993; Horn and Greene, 1995; Horn et al., 1996; ogy is a critical area that needs to be investigated and Papa, 1986). evaluated thoroughly due to the fungal structures ese traditional methods are based upon mor- present serving as the primary cause of a atoxin con- phological and genetic characteristics and are both tamination of agricultural commodities. time consuming and complex (Klich and Pitt 1998; Klich et al., 1992). Recently, additional molecular techniques have been developed to help identify Reducing a atoxin contamination using and classify these fungi (reviewed in Wilkinson and fungicides and herbicides Abbas, 2008). One of the more reliable molecular methods for distinguishing Aspergilli is ribosomal Early investigation in vitro indicates that the fungi- DNA (rDNA) comparisons. Comparisons of variations cide chlobenthiazone is highly eective in inhibit- within 16S rRNA gene sequences of bacterial species ing a atoxin biosynthesis by cultures of A. avus; and 18S rRNA gene sequences for eukaryotes are used however, a atoxin synthesis by A. parasiticus was, extensively for ecological investigations. In particular, in fact, stimulated by this fungicide (Wheeler, 1991). the internal transcribed spacer (ITS) regions are use- Various surfactants, including some used in pesti- ful for identications of fungal species and strains cide formulations, reduced a atoxin biosynthesis by within a species (Hugenholtz and Pace, 1996; White >96% (Rodriguez and Mahoney, 1994). Use of natu- et al., 1990). PCR amplication and sequencing has ral oils from thyme (Kumar et al., 2008), lemongrass also been used for resolving lamentous fungal spe- (Bankole and Joda, 2004), and other herbs has also cies, including A. avus (Accinelli et al., 2008a; Brun been studied and shown to repress a atoxin in cer- et al., 1991; Peterson, 2008; Scully and Bidochka, 2006; tain crops in Asia. White et al., 1990), using comparisons based upon the In the Mississippi Delta, a 3-year eld study has a atoxin biosynthetic pathway, which is unique to the indicated that none of ve fungicides (azoxystrobin, Aspergilli. pyraclostrobin, propiconazole, tetraconazole, e genes involved in the a atoxin biosynthe- dithiocarbamate) or fungicide mixtures of tri oxys- sis pathway have been identied and sequenced trobin + propiconazole and azoxystrobin + propico- (Bhatnagar et al., 2003; Scheidegger and Payne, 2003; nazole, applied to corn at midsilking, were eective in Yu et al., 2004). ese genes are highly conserved in signicantly reducing a atoxin contamination (Gabe multiple Aspergilli, most notably A. parasiticus and Sciumbato, personal communication). A. avus. PCR based on selected genes from the e herbicide glufosinate has been reported as hav- biosynthetic pathway are slowly being adapted for ing antifungal activity against certain phytopathogenic detection, identication, or examination of ecologi- fungi in vitro (Uchimiya et al., 1993) and has shown cal aspects of A. avus. Amplication of these specic activity in reducing infection of corn kernels in vitro genes is extremely sensitive and has the potential to be (Tubajika and Damann, 2002). Much of the corn planted 148 HK Abbas et al. in North America are hybrids resistant to glypho- (Catangui and Berg, 2006; Hesseltine et al., 1976; sate (Roundup Readyv), glufosinate (Liberty Linkv), Lillehoj et al., 1976, 1975, 1980; Wilson et al., 1981). or stacked with resistance to both herbicides. us, a Because environmental factors can greatly aect 4-year study in Mississippi eld trials was performed a atoxin production in preharvest corn, it is not sur- to assess the potential for glufosinate and applications prising to nd that data from a number of investiga- of urea to reduce a atoxin in corn inoculated with A. tions have failed to show any relationship between avus (Bruns and Abbas, 2006). No signicant eect insect damage and the incidence of A. avus or a a- using glufosinate was observed, and foliar applica- toxin (Lillehoj et al., 1980; McMillian et al., 1978, 1980; tion of urea actually increased the level of a atoxin Widstrom et al., 1975). Also, kernels and ear samples contamination in one of the 4 years. have been found that were essentially free of insect Similarly, the incidence of a atoxin in glyphosate- damage, yet contained moderate to high amounts of resistant and conventional corn and cotton hybrids a atoxin (Lee et al., 1980; Rambo et al., 1974; Shotwell was compared in a separate 4-year study (Reddy et al., 1977, Widstrom et al., 1975). e diculty in et al., 2007). Higher levels of a atoxin were observed establishing this relationship is in part due to A. avus in glyphosate-resistant corn compared wih traditional ability to colonize silks, infect kernels, and produce corn hybrids. us, eects of glyphosate on in vitro a atoxins in developing ears under insect-free condi- growth of A. avus in pure culture and on native soil tions (Jones et al., 1980), and in part due to unknown populations were examined, nding that high levels factors that result in con icting information. of glyphosate (>5mM) were required for inhibition. In Windham et al. (1999) reported that a atoxin levels addition, application of several-fold greater than eld were signicantly higher in 1995 when commercial rates of application were found to have no eect on hybrids were both infested with southwestern corn A. avus populations. Interestingly, A. avus, when borer larvae and inoculated with A. avus spores than grown on glyphosate water agar media, produced when they were inoculated with A. avus spores alone. 20% of the a atoxin produced on water agar without In 1996, however, a atoxin contamination was gener- glyphosate. However, despite billions of dollars spent ally lower and was not increased signicantly by infest- annually in fungicide research, little has been gained ing developing ears with southwestern corn borer in identifying products that are active in controlling larvae. ese studies illustrate the need for continued a atoxin contamination. Currently, CIMMYT does research and more thorough examinations of the prob- not recommend foliar application of fungicide to pre- lem in areas where corn production is relatively new. vent a atoxin contamination because of the lack of Because the relationship between insect damage to documented ecacy (CIMMYT, 2008). corn ears and a atoxin is heavily in uenced by envi- ronmental conditions, success in managing a atoxin contamination via insect control has been highly Reducing a atoxin contamination by insect variable. Research has demonstrated that insecti- management cides cannot be applied economically to control corn insects well enough to reduce a atoxin to acceptable A. avus is not considered to be an aggressive invader levels (reviewed in Dowd, 1998). e most success- of preharvest corn ear tissue. However, develop- ful approach has been the use of corn resistant to ing grain when damaged is easily contaminated by ear-feeding insects. Several authors have shown that the pathogen (Diener et al., 1987). e association Bacillus thuringiensis (Bt)–transformed corn hybrids, between insect damage and fungal infection of corn which are resistant to ear-feeding insects, reduce ears was rst recognized when Riley (1882) reported a atoxin contamination of the grain (Dowd, 2000; molds appearing on corn-ear tips soon after being Munkvold et al., 1999; Williams et al., 2002). e adop- infested with insect larvae. Garman and Jewett (1914) tion of Bt corn hybrids has given producers a crop with reported that in years with high insect populations, increased insect resistance, however these hybrids the incidence of moldy ears in eld corn increased. may only reduce a atoxin contamination under cer- Eorts to determine the specic role of insects in the tain circumstances. However, commercial production A. avus infection process increased dramatically of these genetically modied hybrids is not allowed in when a atoxin was recognized as a health concern, some nations. Developing lines with greater natural leading to recognition that ear feeding insects (e.g., insect resistance will be another and perhaps more corn earworm, Helicoverpa zea; European corn borer, acceptable and cost-eective method of reducing Ostrinia nubilalis; fall armyworm, Spodoptera fru- losses to insects and a atoxin. Several sources of giperda; western bean cutworm, Striacosta albicosta; natural resistance to insects have been identied, and and southwestern corn borer, Diatraea grandiosella) crosses between insect- and a atoxin-resistant lines can increase a atoxin levels in preharvest corn have shown potential to increase resistance to both Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 149 insect damage and a atoxin contamination (Williams reduced. Jones et al. (1981) reported a reduction et al., 2002). in A. avus infection and a atoxin contamination in irrigated versus nonirrigated corn and observed these dierences in years of below-normal rainfall. Limiting mycotoxins through crop However, Bruns (2003) did not observe a dierence management in a atoxin or fumonisin (produced by Fusarium verticillioides) contamination levels between irri- Control of mycotoxins in corn, especially a atoxin, by gated and nonirrigated corn. With both mycotoxins genetic manipulation of the host plant has resulted in being observed to have extremely low levels for limited success. e primary means of reducing the all years of the experiment and in both irrigation incidence of contamination remains avoidance of treatments, it is possible that other complicating conditions favorable for infection. Drought and heat environmental factors, such as plant nutrition, were stress during the time that corn matures are the prin- more in uential. ciple contributing factors to a atoxin contamination. Adequate plant nutrition, especially nitrogen, is Such conditions are more common in the southeast- necessary to prevent a atoxin contamination of pre- ern United States, resulting in a atoxin contamina- harvest corn grain. Insucient levels of mineralized tion of corn being more of a problem there than in nitrogen in the root zone, due to either drought stress the rest of the nation (Bruns, 2003). Similarly, in less or leaching due to excessive rain, may predispose corn developed countries where infrastructure and nan- to a atoxin contamination (Jones, 1979). Anderson cial resources are limited, mycotoxin contamination et al. (1975) reported that higher levels of a atoxin of corn grain is also a considerable problem. contamination occurred in corn grain produced with High temperatures are likely the most important 80kg ha−1 of nitrogen compared with grain produced environmental factor in uencing preharvest infec- using 120kg ha−1. Bruns and Abbas (2005a), however, tion of corn by A. avus and the eventual contamina- observed no dierences among irrigated early-season tion of grain by a atoxin (Fortnum, 1986; Jones et al., corn hybrids grown in the mid-South with respect to 1980). As temperatures increase a atoxin levels also a atoxin or fumonisin contamination when nitro- increase (Manwiller and Fortnum, 1979; Williams gen fertilizer levels varied between 112kg ha−1 and et al., 2003). Naturally, nothing can be done to con- 224kg ha−1. Subsequent research at the same location trol ambient temperatures, but it is possible to avoid using a full-season hybrid and varying plant popula- their full impact during the later stages of kernel ll- tions and nitrogen fertility levels produced similar ing by early planting. April plantings of corn in North results (Bruns and Abbas, 2005b). Carolina had lower levels of a atoxin B1 contamina- tion than grain produced by May plantings (Jones and Duncan, 1981; Jones et al., 1981). However, Bruns Conclusions and Abbas (2006) reported that regardless of plant- ing date or maturity rating, corn that experienced 41 It is clear that traditional control approaches, such days of ambient temperatures >32° C during kernel as fungicides, have shown minimal success in man- lling had greater levels of a atoxin contamination aging a atoxin. Fungal biocontrol, insect control, than subsequent years that had 30 days of ambient and novel germplasm appear to hold the most temperatures >32° C. In the southeast United States, promise in mitigating Aspergillus infection and a a- high heat is generally accompanied by low rainfall or toxin contamination. No single technology will be drought. the answer, but these technologies combined with Drought stress can occur in corn rather quickly, more eective crop management will contribute to especially during reproductive growth, if exposed to reducing this important limitation to economical a combination of high ambient temperatures and low corn production. An increased understanding of the relative humidity. In early corn hybrids, yield reduc- relationships between A. avus, the host, the envi- tions of more than 50% could result when brief peri- ronment, and a atoxin is critical to advancing these ods of wilting occurred at 50% silking (Denmead and management strategies and practices. Although Shaw, 1960;Robins and Domingo, 1953). Corn hybrids our knowledge is still incomplete in understanding produced in the 1980s were more tolerant to drought this fungus, advancements in understanding bio- than those from earlier times (Duvick, 1984) and do synthesis and regulation, Aspergilli life cycle, and not appear to suer the adverse aects of drought as environmental eects will continue to increase as soon as older hybrids. additional molecular techniques are developed When corn can be irrigated, drought stress and specically applied to Aspergillus ecology. e may be lowered and a atoxin levels appear to be integration of these approaches in a cost-eective 150 HK Abbas et al. manner may be the greatest challenge we will Angle JS, Dunn KA, Wagner GH. (1982). Eect of cultural practices face in providing safer food and feed production in on the soil population of Aspergillus avus and Aspergillus parasiticus. Soil Sci Soc Am J. 46: 301–304. the 21st century. Bankole SA, Joda AO. (2004). Eect of lemon grass (Cymbopogon citratus Stapf) powder and essential oil on mould dete- rioration and a atoxin contamination of meion seeds Acknowledgments (Colocynthis citrullus L.). Afr J Biotechnol. 3: 52–59. Bayman P, and Cotty PJ. (1993). Genetic diversity in Aspergillus avus: association with a atoxin production and morphol- e authors are grateful to Ms. Lynn Libous-Bailey ogy. Can J Bot. 71: 23–34 and Ms. Bobbie J. Johnson, USDA-ARS, Stoneville, Benndorf D, Műller A, Bock K, Manuwald O, Herbarth O, Van Mississippi, for their technical assistance in this Bergen M. (2008). Identication of spore allergens from the project. is work was approved for publication indoor mold Aspergillus vesicolor. Allergy. 63: 454–460. as Journal Article No. J-11489 of the Mississippi Bennett JW. (1981). Loss of norsolorinic acid and a atoxin production by a mutant of Aspergillus parasiticus. J Gen Agricultural and Forestry Experiment Station, Microbiol. 124: 429–432. Mississippi State University. Bennett JW, Chang PK, Bhatnagar D. (1997). One gene to whole pathway: the role of norsolorinic acid in a atoxin research. Declaration of interest: e authors report no con- Adv Appl Microbiol. 45: 1–15. icts of interest. Bhatnagar D, Cary JW, Ehrlich K, Yu J, Cleveland TE. (2006a). Understanding the genetics of regulation of a atoxin produc- tion and Aspergillus avus development. Mycopathologia. 162: 155–166. Bhatnagar D, Ehrlich KC, Cleveland TE. (2003). Molecular References genetic analysis and regulation of a atoxin biosynthesis. Appl Microbiol Biotechnol. 61: 83–93. Abbas HK, Accinelli C, Zablotowicz RM, Abel CA, Bruns HA, Bhatnagar D, Proctor R, Payne GA, Wilkinson J, Yu J, Cleveland TE, Dong Y, Shier WT. (2008a). Dynamics of mycotoxin and Nierman WC. (2006b). Genomics of mycotoxigenic fungi. In: Aspergillus avus levels in aging Bt and non-Bt corn residues Barug D, Bhatnagar D, van Egmond HP, van der Kamp JW, van under Mississippi no-till conditions. J Agric Food Chem. 56: Osenbruggen WA, Visconti A, eds. e Mycotoxin Factbook 7578–7585. (Food & Feed Topics). Wageningen, e Netherlands: Abbas HK, Weaver MA, Zablotowicz RM, Horn BW, Shier WT. Wageningen Academic Publishers, pp. 157–178. (2005). Relationships between a atoxin production, sclero- Bhatnagar D, Yu J, Ehrlich KC. (2002). Toxins of lamentous fungi. tia formation and source among Mississippi Delta Aspergillus Chem Immunol. 81: 167–206. isolates. Eur J Plant Pathol. 112: 283–287. Blout WP. (1961). Turkey “X” disease. Turkeys. 52: 55–58. Abbas HK, Zablotowicz RM. Bruns HA. (2008b). Modelling Brun TD, White TJ, Taylor JW. (1991). Fungal molecular systemat- the colonization of maize by toxigenic and non-toxigenic ics. Annual Review of Ecological System. 22: 525–564. Aspergillus avus strains: implications for biological control. Bruns HA. (2003). Controlling a atoxin and fumonisin in maize World Mycotoxin Journal. 1: 333–340. by crop management. J Toxicol Toxin Rev. 22: 153–173. Abbas HK, Zablotowicz RM, Bruns HA, Abel CA. (2006). Biocontrol Bruns HA, Abbas HK. (2005a). Response of short-season corn of a atoxin in corn by inoculation with non-a atoxigenic hybrids to a humid sub-tropical environment. Agron J. 97: Aspergillus avus isolates. Biocontrol Sci Technol. 16: 437–449. 446–451. Abbas, H. K., Zablotowicz, R. M., Bruns, H. A., and Abel., C. A. Bruns HA, Abbas HK. (2005b). Ultra-high plant populations and (2008c). Development of non-toxigenic strains of Aspergillus N-fertility eects on corn in the Mississippi Valley. Agron J. avus for control of a atoxin in Maize. In: Burton EN, 97: 1136–1140. Williams PV, eds. Crop Protection, Research Advances. New Bruns HA, Abbas HK. (2006). Eects of glufosinate – Ammonium York, NY: Nova Sciences Publishers, pp. 181–192. and urea on a atoxin and fumonisin levels in corn. Plant Abbas HK, Zablotowicz RM, Locke MA. (2004a). Spatial vari- Health Progress. doi:10.1094/PHP-2006-0324-01-RS. ability of Aspergillus avus soil populations under dierent Campbell BC, Molyneaux RJ, Schatza BS. (2003) Current research crops and corn grain colonization and a atoxins. Can J Bot. on reducing pre- and post-harvest a atoxin contamination 82: 1768–1775. of U.S. almond, pistachio, and walnut. J Toxicol Toxin Rev. Abbas HK, Zablotowicz RM, Weaver MA, Horn BW, Xie W, Shier WT. 22: 225–266. (2004b). Comparison of cultural and analytical methods for Cary JW, Calvo AM. (2008). Regulation of Aspergillus mycotoxin determination of a atoxin production by Mississippi Delta biosynthesis Toxin Reviews 27 (3 & 4) 347–370. Aspergillus isolates. Can J Microbiol. 50: 193–199. Cary JW, Ehrlich K. (2006). A atoxigenicity in Aspergillus: molec- Accinelli C, Abbas HK, Zablotowicz RM, Wilkinson JR. (2008a). ular genetics, phylogenetic relationships and evolutionary Aspergillus avus a atoxin occurrence and expression of a a- implications. Mycopathologia. 162: 167–177. toxin biosynthesis genes in soil. Can J Microbiol. 54: 371–379. Catangui MA, Berg RK. (2006). Western bean cutworm, Striacosta Accinelli C, Abbas HK, Zablotowicz RM, Wilkinson JR. (2008b). albicosta (Smith) (Lepidoptera: Noctuidae), as a potential Role of Ostrinia nubilalis in vectoring Aspergillus avus pest of transgenic Cry1Ab Bacillus thuringiensis corn hybrids in a corn eld in northern Italy. Proceedings of the 235th in South Dakota. Environ Entomol. 35: 1439–1452. American Chemical Society (ACS) National Meeting, April CIMMYT. (2008). Aspergillus ear rot (extended information). 6–10, New Orleans, LA. http://maizedoctor.cimmyt.org/index.php?option=com_ Anderson HW, Nehring EW, Wichser WR. (1975). A atoxin content&task=view&id=227. Accessed October 28, 2008. contamination of corn in the eld. J Agric Food Chem. 23: Cleveland TE, Dowd PF, Desjardins AE, Bhatnagar D, Cotty, PJ. 775–782. (2003). United States Department of Agriculture-Agricultural Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 151

Research Service research on pre-harvest prevention of Heathcote JG, Hibbert JR. (1978). A atoxins: Chemical and mycotoxins and mycotoxigenic fungi in US crops. Pest Biological Aspects. Amsterdam, e Netherlands: Elsevier Manag Sci. 59: 629–642. Scientic Publishing Company. Cotty PJ. (1994). In uence of eld application of an atoxigenic Hesseltine CW, Shotwell OL, Kwolek WF, Lillehoj EB, Jackson WK, strain of Aspergillus avus on the populations of A. avus Bothast R.J., (1976). A atoxin occurrence in 1973 corn at har- infecting cotton bolls and on the a atoxin content of cotton- vest. II. Mycological studies. Mycologia. 68: 341–353. seed. Phytopathology. 84: 1270–1277. Hill RA, Wilson DM, McMillian WW, Widstrom NW, Cole RJ, Cotty PJ, Antilla L, Wakelyn PJ. (2007). Competitive exclusion of Sanders TH, Blankenship PD. (1985). Ecology of the Aspergillus a atoxin producers: farmer driven research and develop- avus group and a atoxin formation in maize and ground- ment. In: Vincent C, Goettel N, Lazarovitis G, eds. Biological nut. In: Lacey J, ed. Trichothecenes and Other Mycotoxins. Control: A Global Perspective. Oxfordshire, UK: CAB Chichester, UK: John Wiley & Sons, pp. 79–95. International, pp. 241–253. Horn BW. (2003). Ecology and population biology of a atoxigenic Cotty PJ, Bayman P. (1993). Competitive exclusion of a toxi- fungi in soil. J Toxicol Toxin Rev. 22: 355–383. genic strain of Aspergillus avus by an atoxigenic strain. Horn BW. (2007). Biodiversity of Aspergillus section Flavi in the Phytopathology. 83: 1283–1287. United States: a review. Food Addit Contam. 24: 1088–1101. Criseo G, Bagnara A, Bisignano G. (2001). Dierentiation of a a- Horn BW, Dorner JW. (1998). Soil populations of Aspergillus toxin-producing and non-producing strains of Aspergillus species from section Flavi along a transect through pea- avus group. Lett Appl Microbiol. 33: 291–295. nut-growing regions of the United States. Mycologia. 90: Das MK, Ehrlich KC, Cotty PJ. (2008). Use of pyrosequencing 767–776. to quantify incidence of a specic Aspergillus avus strain Horn BW, Greene RL, Dorner JW. (1995). Eect of corn and within complex fungal communities associated with com- peanut cultivation on soil populations of Aspergillus avus mercial cotton crops. Phytopathology. 98: 282–288. and A. parasiticus in southwestern Georgia. Appl Environ Degola F, Berni E, Dall’Asta C, Spotti E, Marchelli R, Ferrero I, Microbiol. 61: 2472–2475. Restivo FM. (2006). A multiplex RT-PCR approach to detect Horn BW, Greene RL, Sobolev S, Dorner JW, Powell JH, a atoxigenic strains of Aspergillus avus. J Appl Microbiol. Layton RC. (1996). Association of morphology and myco- 103: 409–417. toxin production with vegetative compatibility groups in Denmead OT, Shaw RH. (1960). e eects of soil moisture stress Aspergillus avus, A. parasiticus, and A. tamarii. Mycologia. at dierent stages of growth on the development and yield of 88: 574–587. corn. Agron J. 52: 272–274. Hugenholtz P and NR Pace. (1996) A molecular phylogenetic Diener UL, Cole RJ, Sanders TH, Payne GA, Lee LS, Klich MA. approach to the natural microbial world: the biotechnologi- (1987). Epidemiology of a atoxin formation by Aspergillus cal potential. Trends Biotechnol. 14:190–197. avus. Annu Rev Phytopathol. 25: 249–270. Jaime-Garcia R, Cotty PJ. (2004). Aspergillus avus in soils Dorner JW. (2008). Management and prevention of mycotoxins and corncobs in South Texas: Implications for manage- in peanuts. Food Addit Contam. 25: 203–208. ment of a atoxins in corn-cotton rotations. Plant Dis. 88: Dorner JW, Cole RJ, Daigle DJ, McGuire MR, Shasha BS. (2003) 1366–1371. Evaluation of biological control formulations to reduce Jones RK. (1979). Epidemiology and management of a atoxins a atoxin contamination in peanuts. Biol Control. 26: and other mycotoxins. In: Harsfall, JG, Cowling EB, eds. 318–324. Plant Diseases: An Advanced Treatise. Vol. IV. New York, NY: Dorner JW, Cole RJ, Wicklow DT. (1999). A atoxin reduction in Academic Press, p. 466. corn through eld application of competitive fungi. J Food Jones RK, Duncan HE. (1981). Eect of nitrogen fertilizer, plant- Protect. 62: 650–656. ing date, and harvest date on a atoxin production in corn Dowd PF. (1998). e involvement of arthropods in the estab- inoculated with Aspergillus avus. Plant Dis. 65: 741–744. lishment of mycotoxigenic fungi under eld conditions. In: Jones RK, Duncan HE, Payne GA, Hamilton PB. (1981). Planting Sinha KK, Bhatnagar D, eds. Mycotoxins in Agriculture and date, harvest date, and irrigation eects on infection and Food Safety. New York, NY: Marcel Dekker, pp. 307–350. a atoxin production by Aspergillus avus in eld corn. Dowd PF. (2000). Indirect reduction of ear molds and associated Phytopathology. 66: 675–677. mycotoxins in Bacillus thuringiensis in corn under control- Jones RK, Duncan HE, Payne GA, Leonard KJ. (1980). Factors led and open eld conditions: utility and limitations. J Econ in uencing infection by Aspergillus avus in silk-inoculated Entomol. 93: 1669–1679. corn. Plant Dis. 64: 859–863. Duvick DN. (1984). Genetic contribution to yield gains of U.S. Klich MA. (1986). Myco ora of cotton seed from the southern hybrid maize, 1930 to 1980. In: Genetic Contributions to United States: a three year study of distribution and fre- Yield Gains of Five Major Crop Plants. Madison, WI: ASA, quency. Mycologia. 78: 706–712. CSSA, pp. 15–47. Klich MA. (2002). Biogeography of Aspergillus species in soil and Fortnum BA. (1986). Eect of environment on a atoxin develop- litter. Mycologia 94: 21–27. ment in preharvest maize. In: Zuber MS, Lillehoj EB, eds. Klich MA. (2007). Aspergillus avus: the major producer of a a- A atoxin in Maize: A Proceedings of the Workshop. El Batan, toxin. Mol Plant Pathol. 8: 713–722. Mexico: CIMMYT, pp. 145–151. Klich MA, Pitt JI. (1988). Dierentiation of Aspergillus avus from Garman H, Jewett HH. (1914). e life-history and habits of the Aspergillus parasiticus and other closely related species. corn-ear worm (Chloridea obsoleta). Kentucky Agricultural Trans Br Mycol Soc. 91: 99–108. Experimental Station Bulletin. 187: 513–591. Klich MA, Tiany LH, Knaphus G. (1992). Ecology of the Geisen R. (1996). Multiplex polymerase chain reaction for the Aspergilli of soils and litter. In: Bennett JW, Klich MA, eds. detection of potential a atoxin and sterigmatocystin pro- Aspergillus: Biology and Industrial Applications. Boston, ducing fungi. Syst Appl Microbiol. 19: 388–392. MA: Butterworth-Heinemann, pp. 329–353. Grin GJ, Garren KH, Taylor JD. (1981). In uence of crop rota- Kumar A, Shukla R, Singh P, Prasad CS, Dubey NK. (2008). tion and minimum tillage on the populations of Aspergillus Assessment of ymus vulgaris L. essential oil as a safe avus group in peanut eld soil. Plant Dis. 65: 898–900. botanical preservative against post harvest fungal infestation 152 HK Abbas et al.

of food commodities. Innovative Food Science and Emerging propagules in a corn and cotton glyphosate-resistant crop- Technologies. 9: 575–580. ping systems. Food Addit Contam. 24(12): 1367–1373. Lee LS, Lillehoj EB, Kwolek WF. (1980). A atoxin distribution in Riley CV. (1882). e boll-worm alias corn-worm (Heliothis individual corn kernels from intact ears. Cereal Chem. 57: armigera Hübn.) order Lepidoptera; family Noctuidae. 340–343. In: Report of the Commissioner of Agriculture for the years Lillehoj EB, Fennell DI, Kwolek WF. (1976). Aspergillus avus 1881 - 1882. Washington Printing Oce, pp. 145–152. and a atoxin in Iowa corn before harvest. Science. 193: Robins JS, Domingo CE. (1953). Some eects of severe soil moisture 495–496. decits at specic growth stages in corn. Agron J. 45: 618–621. Lillehoj EB, Kwolek WF, Horner ES, Widstrom NW, Josephson LM, Rodriguez SB, Mahoney NE. (1994). Inhibition of a atoxin pro- Franz AO, Catalano A. (1980). A atoxin contamination of pre- duction by surfactants. Appl Environ Microbiol. 60: 106–110. harvest corn: Role of Aspergillus avus inoculum and insect Scheidegger KA, Payne GA. (2003). Unlocking the secrets behind damage. Cereal Chem. 57: 255–257. secondary metabolism: a review of Aspergillus avus from Lillehoj EB, Kwolek WF, Fennell DI, Milburn MS. (1975). A atoxin pathogenicity to functional genomics. J Toxicol Toxin Rev. incldence association with bright greenish-yellow uores- 22: 423–459. cence and insect damage in a limited survey of freshly har- Scherm B, Palomba M, Serra D, Marcello A, Migheli Q. (2005). vested high-moisture corn. Cereal Chem. 52: 403–412. Detection of transcripts of the a atoxin genes a D, a O, Lyn ME, Abbas HK, Zablotowicz RM. Johnson, BJ. (2009). and a P by reverse transcription-polymerase chain reaction Delivery systems for biological control agents to manage allows dierentiation of a atoxin-producing and non-pro- a atoxin contamination of pre-harvest maize. Food Addit ducing isolates of Aspergillus avus and Aspergillus parasiti- Contam. cus. Int J Food Microbiol. 98: 201– 210. Manonmani HK, Anand S, Chandrashekar A, Rati, ER. (2005). Scully LR, Bidochka MJ. (2006). A cysteine/methionine auxotroph Detection of atoxigenic fungi in selected food commodities of the opportunistic fungus Aspergillus avus is associated by PCR. Process Biochemistry 40: 2859–2864. with host-range restriction: a model for emerging diseases. Manwiller A, Fortnum B. (1979). A comparison of a atoxin levels Microbiology. 152: 223–232. on pre-harvest corn from the South Carolina coastal plain Sharnma D, Dutta DK, Singh AB, Shome BR. (2007). for 1977–78. Agron. and Soils Research. Series 201. South Aerobiological, biochemical and immunological studies on Carolina Agric. Exp. Stn., Florence, SC. some of the dominant Aspergillus species of South Assam McMillian WW, Wilson DM, Widstrom NW. (1978). Insect (India). Aerobiologia. 23: 201–210. damage, Aspergillus avus ear mold, and a atoxin contami- Shotwell OL, Goulden ML, Lillehoj EB, Kwolek WF, Hesseltine CW. nation in south Georgia corn elds in 1977. J Environ Qual. (1977). A atoxin occurrence in 1973 corn at harvest III. 7: 564–566. A atoxin distribution in contaminated, insect-damaged McMillian WW, Wilson DM, Widstrom NW, Gueldner RC. (1980). corn. Cereal Chem. 54: 620–626. Incidence and level of a atoxin in pre-harvest corn (Zea Smith CA, Woloshuk CP, Robertson D, Payne GA. (2007) Silencing mays) in south Georgia, USA in 1978. Cereal Chem. 57: of the a atoxin gene cluster in a diploid strain of Aspergillus 83–84. avus is suppressed by ectopic a R expression. Genetics. Minto RE, Townsend CA. (1997). Enzymology and molecular 176: 2077–2086. biology of a atoxin biosynthesis. Chem Rev. 97: 2537–2556. Teertstra WR, Lugones LG, Wosten HAB. (2004). In situ hybridiza- Munkvold GP, Hellmich RL, Rice LG. (1999). Comparison of tion in lamenous fungi using peptide nucleic acid probes. fumonisin concentrations in kernels of transgenic Bt maize Fungal Genet Biol. 41: 1099–1103. hybrids and non-transgenic hybrids. Plant Dis. 83: 130–138. Townsend CA. (1997). Progress towards a biosynthetic rationale O’Brian GR, Georgianna DR, Wilkinson JR, Abbas HK, Wu J, of the a atoxin pathway. Pure Appl Chem. 58: 227–238. Bhatnagar D, Cleveland TE, Nierman W, Payne GA. (2007). Tubajika KM, Damann KE Jr. (2002). Glufosinate-ammonium e eect of elevated temperature on gene expression and reduces growth and a atoxin B1 production by Aspergillus a atoxin biosynthesis. Mycologia. 90: 232–239. avus. J Food Prot. 65: 1483–1487. Papa KE. (1986). Heterokaryon incompatibility in Aspergillus Uchimiya H, Iwate M, Nojiri C, Samarajeewa PK, Takamatsu S, avus. Mycologia. 78: 98–101. Ooba S, Anzai H, Christensen AH, Quail PH, Toki S. (1993). Payne GA. (1992). A atoxin in maize. CRC Crit Rev Plant Sci. 10: Bialaphos treatment of transgenic rice plants expressing a 423–440. bar gene prevents infection by the sheath blight pathogen Payne GA. (1998). Process of contamination by a atoxin- (Rhizoctonia solani). Biotechnology 11: 835–836. producing fungi and their impacts on crops. In: Sinha KK, Wheeler MH. (1991). Eects of chlobenthiazone on a atoxin bio- Bhatnagar D, eds. Mycotoxins in Agriculture and Food synthesis in Aspergillus parasiticus and A. avus. Pesticide Safety, Vol. 9. New York, NY: Marcel Dekker, pp. 279–306. Biochemistry and Physiology 41: 190–197. Payne GA, Brown MP. (1998). Genetics and physiology of a atoxin White TJ, Bruns TD, Lee S, Taylor JW. (1990). Amplication of biosynthesis. Annu Rev Phytopathol. 36: 329–362. direct sequencing of fungal ribosomal RNA genes for phy- Peterson SW. (2008). Phylogenetic analysis of Aspergillus species logenetics. Innis MA, Gelfand DH, Sninsky JJ, eds. In: PCR using DNA sequences from four loci. Mycologia. 100: Protocols: A Guide to Methods and Applications New York, 205–226. NY: Academic Press, Inc., pp. 315–322. Pildain MB, Vaamonde, G, Cabral D. (2004). Analysis of popu- Wicklow DT, Mcalpin CE, Platis CE. (1998). Characterization of lation structure of Aspergillus avus from peanut based on the Aspergillus avus population within an Illinois maize vegetative compatibility, geographic origin, mycotoxin and eld. Mycol Res. 102: 263–268. sclerotia production. Int J Food Microbiol. 93: 31–40. Wicklow DT, Wilson DM, Nelsen TE. (1993). Survival of Aspergillus Rambo GW, Tuite J, Crane P. (1974). Pre-harvest inoculation and avus sclerotia and conidia buried in soil in Illinois and infection of dent corn with Aspergillus avus and Aspergillus Georgia. Phytopathology. 83: 1141–1147. parasiticus. Phytopathology. 64: 797–800. Wicklow DT, Bobell JR, Palmquist DE. (2003). Eect of intraspe- Reddy KN, Abbas HK, Zablotowicz RM, Abel CA, Kroger CH. cic competition by Aspergillus avus on a atoxin formation (2007). Mycotoxin occurrence and Aspergillus avus soil in suspended disc culture. Mycol Res. 107: 617–623. Ecology of Aspergillus avus, regulation of a atoxin production, and management strategies 153

Widstrom NW, Sparks AN Lillehoj EB, Kwolek FW. (1975). Biosynthesis, Genetics and Regulation. Trivandrum, India: A atoxin production and lepidopteran insect injury on corn Research Signpost, pp. 227–251. in Georgia. J Econ Entomol. 68: 855–856. Yu J. (2004). Genetics and biochemistry of mycotoxin synthe- Wilkinson JR, Abbas HK. (2008). A atoxin, Aspergillus, maize, and sis. In: Arora DK, ed. Fungal Biotechnology in Agricultural, the relevance to alternative Fuels (or A atoxin: What is it, can Food, and Environmental Applications. New York, NY: we get rid of it, and should the ethanol industry care?). Toxin Marcel Dekker, Inc., pp. 343–361. Rev. 27 (3&4) 227–260. Yu J, Bhatnagar D, Ehrlich KC. (2002). A atoxin biosynthesis. Rev Williams WP, Buckley PM, Windham GL. (2002). Southwestern Iberoam Micol. 19: 191–200. corn borer (Lepidoptera: Crambidae) damage and a atoxin Yu J, Chang P-K, Cary J, Wright WM, Bhatnagar D, Cleveland TE, accumulation in maize. J Econ Entomol. 95: 1049–1053. Payne GA, Linz JE. (1995). Comparative mapping of a a- Williams WP, Windham GL, Buckley PM. (2003). A atoxin accu- toxin pathway gene clusters in Aspergillus parasiticus and mulation in maize after inoculation with Aspergillus avus Aspergillus avus. Appl Environ Microbiol. 61: 2365–2371. and infestation with southwestern corn borer. J Genet Breed. Yu J, Chang PK, Ehrlich KC, Cary JW, Bhatnagar D, Cleveland 57: 365–369. TE, Payne GA, Linz JE, Woloshuk CP, Bennett JW. (2004). Wilson DM, Widstrom, NW, Marti LR, Evans BD. (1981). Clustered pathway genes in a atoxin biosynthesis. Appl Aspergillus avus group, a atoxin, and bright greenish-yel- Environ Microbiol. 70: 1253–1262. low uorescence in insect-damaged corn in Georgia. Cereal Yu J, Cleveland TE, Nierman WC, Bennett JW. (2005). Aspergillus Chem. 58: 40–42. avus genomics: gateway to human and animal health, food Windham GL, Williams WP, Davis FM. (1999). Eects of the south- safety, and crop resistance to diseases. Rev Iberoam Micol. ern corn borer on Aspergillus avus kernel infection and a a- 22: 194–202. toxin accumulation in maize hybrids. Plant Dis. 83: 535–540. Zablotowicz RM, Abbas HK, Locke MA. (2007). Population ecol- Yabe K. (2003). Pathway and genes of a atoxin biosynthesis. In: ogy of Aspergillus avus associated with Mississippi Delta Fierro F, Francisco J, eds. Microbial Secondary Metabolites: soils. Food Addit Contam. 24: 1102–1108.