Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2019
Interactions between Aspergillus flavus and stored-grain insects in conventional and transgenic maize
Julie Aiza L. Mandap Iowa State University
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Recommended Citation Mandap, Julie Aiza L., "Interactions between Aspergillus flavus and stored-grain insects in conventional and transgenic maize" (2019). Graduate Theses and Dissertations. 17738. https://lib.dr.iastate.edu/etd/17738
This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Interactions between Aspergillus flavus and stored-grain insects in conventional and transgenic maize
by
Julie Aiza L. Mandap
A thesis submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Major: Plant Pathology
Program of Study Committee Gary P. Munkvold, Major Professor Silvina L. Arias Richard L. Hellmich Dirk E. Maier
The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2019
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TABLE OF CONTENTS
Page
LIST OF FIGURES…………………………………………………………………………………… iii
LIST OF TABLES……………………………………………………………………………………. iv
ABSTRACT………………………………………………………………………………………………. v
CHAPTER 1. GENERAL INTRODUCTION…………………………………………………… 1
Thesis Organization…………………………………………………………………...... 1 Literature Review………………………………………………………………………… 1 Overview and Objectives……………………………………………………………… 29 Literature Cited…………………………………………………………………………… 30
CHAPTER 2. INTERACTIONS BETWEEN ASPERGILLUS FLAVUS AND STORED-GRAIN INSECTS IN CONVENTIONAL AND TRANSGENIC MAIZE…... 48
Abstract………………………………………………………………………………………. 48 Introduction………………………………………………………………………………... 49 Materials and Methods………………………………………………………………… 52 Results………………………………………………………………………………………... 58 Discussion…………………………………………………………………………………… 68 References…………………………………………………………………………………... 75 Figures and Tables………………………………………………………………………. 80
CHAPTER 3. GENERAL CONCLUSIONS.……………………………………………………. 91
iii
LIST OF FIGURES
Page
Figure 1 Preliminary experiment on IMM with and without light……………………. 81
Figure 2 Preliminary experiment on storage at 32°C and 70-75% RH……………… 81
Figure 3 Preliminary experiment on storage at 32°C and 80-75% RH………………
Figure 4 Preliminary A. flavus levels…………………………………….……………………….... 82
Figure 5 IMM colonies on different non-Bt hybrids………………………………………… 83
Figure 6 Feeding damage caused by OK vs KS IMM………………………………………… 83
Figure 7 Indianmeal moth stages and damage………………………………………………... 84
Figure 8 Maize weevil stages and damage……………………………………………………… 84
Figure 9 Background levels of the test hybrids………………………………………………. 85
Figure 10 Survivorship of IMM and maize weevil in non-Bt R19………………………. 86
Figure 11 Mortality and growth index of IMM and MW……………………………………. 87
Figure12 Damage and grain weight loss…………………………………………………………. 88
Figure 13 A. flavus levels (CFU/g of milled grain) ……………………………………………. 89
Figure 14 Photos of A. flavus colony counts on AFPA.……………...………………………… 89
Figure 15 Aflatoxin contamination levels ………………………………………………………... 90
iv
LIST OF TABLES
Page
Table 1 List of maize hybrids used in this study……………………………………………. 80
Table 2 List of Bt proteins evaluated and details of each protein expressed……. 80
Table 3 Background mycotoxin levels of the test hybrids ……………………………… 85
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ACKNOWLEDGEMENTS
I wish to extend my sincerest gratitude to my major professor, Gary Munkvold, for providing me with this opportunity, for the unrelenting support throughout my program, and for the exceptional mentorship. I would also like to thank my committee members
Silvina Arias, Richard Hellmich and Dirk Maier for their guidance. I’m so grateful to the
Seed Pathology Lab members, Gabriela Morel, Fernando Marcos, Tracy Bruns, Charlie
Block, especially Derrick Mayfield, for all their help in the lab and research bits of advice.
Special thanks to Allan Gaul, Dai Nguyen, and Princess Mae Borja for all their assistance and support. I owe a massive thank you to the Fulbright program, the Philippine-American
Educational Foundation, the Commission on Higher Education, and the University of the
Philippines Los Baños for funding and investing their trust on me to do graduate studies here in the US. Finally, a big thanks to my ever-supportive family, friends, and loved ones.
To God be the Glory!
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ABSTRACT
Grain quality of maize after harvest is reduced primarily by mold and insect infestations. If grain temperature and moisture conditions are not controlled, Aspergillus flavus colonization and associated aflatoxin contamination can increase. Indianmeal moth
(Plodia interpunctella Hübner) and maize weevil (Sitophilus zeamais Motschulsky), stored- grain pests in the Order Lepidoptera and Coleoptera, respectively, directly feed on kernels thereby predispose grain to fungal colonization and mycotoxin contamination. The presence of Bt proteins in maize, as a result of deterred insect activity, has been shown to indirectly reduce mycotoxin contamination in the field. Insect-mold interactions have been studied thoroughly in the field but not in storage, particularly the effect of lepidopteran events on Indianmeal moth and coleopteran events on maize weevil. Preliminary experiments were conducted to optimize Indianmeal moth infestation methods. Storage conditions were established based on temperature and humidity ranges common in the tropics, which would also be conducive to A. flavus growth, aflatoxin production, and insect development. The main experiments were then performed using Bt and non-Bt maize hybrids conditioned to 16-17% moisture content. The grains were artificially inoculated with A. flavus at two different levels of inoculum, i.e., 106 and 105 spores/ml. Indianmeal moth and maize weevil were infested into the grains in separate jars with and without A. flavus inoculation. After 28 days of storage, no Indianmeal moth or maize weevil survived in the transgenic hybrids with lepidopteran and coleopteran events, respectively. A. flavus caused increased mortality, reduced survivorship, and growth indices of both Indianmeal moth and maize weevil. Hence, percent grain damage and weight loss were higher in the uninoculated grain because of greater insect feeding than the inoculated grain. Colonization vii of grain by A. flavus was not significantly different between treatments with or without insect infestation, likely because A. flavus suppressed insect activity at these inoculum levels. A. flavus-insect interactions were influenced by the presence of Bt proteins in the maize grain. Insect infestations increased levels of aflatoxin contamination in the non-Bt hybrid inoculated with 106 spores/ml, but did not affect aflatoxin levels in Bt hybrids.
These results indicate that Bt protection against storage insects can reduce the risk of high levels of aflatoxins in grain.
1
CHAPTER 1. GENERAL INTRODUCTION
Thesis Organization
This thesis is composed of an abstract followed by three chapters. The first chapter provides the significance of this study and a literature review of maize, insects, fungi, and mycotoxins with a focus on conventional and transgenic maize, Aspergillus flavus and two stored-grain insects, Indianmeal moth (Plodia interpunctella Hübner) and maize weevil
(Sitophilus zeamais Motschulsky). The second chapter contains protocol development, preliminary and main experimental results, and discussion. The third chapter provides a general summary of the research.
Literature Review
Conventional and transgenic maize as grain
In global cereal grain production, maize (Zea mays L.) comes third after wheat and rice. Maize production has increased for the past decade (Golob et al., 2004) with maize and soybean accounting for an average of 55% of seed expenditures in the United States
(US) from 1996 to 2006 (Roucan-Kane & Gray, 2009). The US has led the world in maize production since 1961 up to present. In 2017, US was again the highest producer of maize with 371M tons, followed by China, and Brazil with 259M and 98M tons, respectively
(FAOSTAT). Maize is a major staple food grain throughout the world, particularly in Sub-
Saharan Africa, Latin America, and Asia (Smale et al., 2011). 2
Maize is mainly used for human food consumption in developing countries. It is also used as feedstock and as raw material in many value-added products such as bioethanol, sorbitol, dextrose, glucose, and oil. Agronomic management of maize-grain fields and seed- crop fields is similar, but the latter is more intensive because of its higher value (Beck,
2002). Maize seed is produced as a hybrid crop and is stored separately from grain in industrialized countries; however, in most developing countries, grain and seed are not clearly distinguished from each other and they are stored together (Pingali & Pandey,
2001). The main objective in grain storage is to minimize economic losses by preserving grain quality and meeting nutritional and product safety standards for both marketing and processing (Chakraverty et al., 2003; Hodges, 2013).
Molds and stored insect pests are a huge problem in grain storage. High temperature and high moisture conditions lead to grain with high moisture content, which are favorable for insect pest development and mold colonization. Grain is ideally dried to a moisture level that suppresses the growth of nearly all storage fungi; however, there are also fungi that can grow at low relative humidity, and many insects also are adapted to survive in dry conditions (Mills, 1982). For example, although maize weevil (Sitophilus zeamais) oviposit during the seed ripening stage, larvae may start development during storage when seed moisture content (MC) is 10% or higher (Howe, 1972). The safe moisture levels for maize grain storage are below 13.5%, but in areas that lack artificial drying facilities and that have limited sunshine hours for drying, harvested grain often is stored at moisture levels between 15-21% (Wilson & Desmarchelier, 1994). Maize grain can be stored safely at a temperature of 18oC or less, but temperatures between 21-27oC are problematic because the majority of storage molds readily grow under these 3 conditions. According to Sauer et al., (1992), relative humidity of the interseed air in storage, rather than the grain MC, is used to estimate the potential for fungal growth; however, the relative humidity of the interseed air is directly related to the grain MC.
Physical or abiotic conditions can be altered to manage storage problems, but maintaining ideal temperature and MC remains a challenge in most storage facilities in developing countries. Importantly, susceptibility of grain to mold colonization and insect infestation also varies across maize hybrids because of differences in physiochemical properties
(Moreno-Martinez & Christensen, 1971; Friday et al., 1989).
Bacillus thuringiensis (Bt) is a spore-forming bacterium that synthesizes crystalline inclusions containing Cry proteins, which are endotoxins that are toxic against a wide range of insects (Palma et al., 2014). The insecticidal properties of Bt proteins were first reported in 1901 by Shigetane Ishiwatari (Ishiwatari, 1901). The first Bt maize hybrids expressing Cry genes encoding for Cry protein production in specific plant tissues were commercially introduced in 1996 (Schnepf et al., 1998). Bt maize hybrids may have single or multiple insect-resistance genes. Hybrids that have multiple Bt-derived proteins targeting a pest, called stacked or pyramided hybrids, confer resistance against a broader spectrum of insect pests compared to those with a single protein. Most of the commercially available hybrids are stacked with herbicide tolerance genes, such as GA21, for glyphosate resistance (Halpin, 2005; Bowers et al., 2013). Bt maize is now the second the most cultivated transgenic crop worldwide next to soybean. Bt maize has been planted in more than 13 countries, i.e., US, Argentina, Canada, South Africa, Philippines, Spain, Uruguay,
Honduras, Portugal, Germany, France, the Czech Republic, and Slovakia. The adoption of stacked traits with Bt genes and herbicide tolerance is most prevalent in the US and Brazil. 4
In 2017, the total area in the US cultivated with transgenic maize is 33.84 million hectares with 1.1 million hectares planted with insect-resistant varieties and 28.34 million hectares with stacked varieties with insect resistance and herbicide tolerance. In Brazil, the total area of maize cultivated with insect resistance was 3.26 million hectares and with stacked varieties was 11.32 million hectares in 2017 (ISAAA, 2017).
Lepidopteran resistance genes
Most primary insect pests attacking maize ears belong to the order Lepidoptera
(Dowd, 2003). Bt maize was primarily created to combat Ostrinia nubilalis Hübner, the
European corn borer (ECB), a lepidopteran pest once regarded as the most widespread insect pest of maize. Cry proteins are beta-endotoxins expressed by B. thuringiensis during sporulation (Estruch et al., 1996). These proteins are toxic to most lepidopteran field pests generally by causing damage to the midgut lining. Cry1Ab protein has been reported effective in reducing ECB feeding (Koziel et al., 1993; Armstrong et al., 1995; Dowd, 2000), but is not as effective against other Lepidopterans such as corn earworm (CEW)
(Helicoverpa zea Boddie) and western bean cutworm (WBC) (Striacosta albicosta Smith)
(Bowers et al., 2014). However, Cry1Ab in combination with Vip3Aa20 is remarkably effective against both insects (Burkness et al., 2010; Bowers et al., 2013). Maize hybrids with events Bt11 or MON810 expressing Cry1Ab protein were reported to be effective against ECB, Asian corn borer (ACB) (Ostrinia furnacalis Guenée), sugarcane borer (SB)
(Diatraea saccharalis Fabricius), southwestern corn borer (SWCB) (D. grandiosella Dyar), mediterranean corn borer (MCB) (Sesamia nonagrioides Cérisy), and spotted stalk/stem borer (SSB) (Chilo partellus Swinhoe) (EPA, 2011). 5
On the other hand, Cry1Fa2 is a modified Cry1F protein derived from B. thuringiensis var. aizawai that works against WBC, black cutworm (BCW) (Agrotis ipsilon
Hufnagel), fall armyworm (FAW) (Spodoptera frugiperda Smith), SWCB, and is mildly effective against CEW (Buntin, 2008; Hardke et al., 2011). The maize event TC1507 expresses Cry1Fa2 was first planted commercially in the US and Argentina in 2001 and
2005, respectively (Baktavachalam et al., 2015). In a multi-site trial in the Philippines,
TC1507 was also shown to confer resistance to ACB (Thompson et al., 2010). In the study of Bowers et al. (2014), Cry1F hybrids were observed to consistently mitigate WBC infestations compared to Cry1Ab or non-Bt hybrids.
Vip3A, unlike the Cry proteins, is not a beta-endotoxin and is produced during the vegetative stage of B. thuringiensis growth beginning at the mid-log phase until sporulation
(Estruch et al., 1996). Vip3Aa20 protein is expressed in event Mir162 that has been evaluated to effectively control CEW, WBC, FAW, and BCW; significantly better than Bt11 maize alone (Yu et al., 1997; EPA, 2009; Burkness et al., 2010). In a field experiment conducted in Minnesota and Maryland, comparison of Vip3A x Cry1Ab hybrids (Mir162 and
Bt11 events) and non-Bt isolines for four growing seasons showed an average of 43-100% ears infested by CEW in the non-Bt and no CEW larvae and damage in Vip3A x Cry1Ab. In hybrids with only Cry1Ab, moderate CEW feeding was observed and surviving larvae were mostly limited to the first and second instar only (Burkness et al., 2010). However, a hybrid that has Mir162 alone, i.e., not pyramided with Bt11 or Mir604 (event expressing mCry3A for coleopteran resistance), is not allowed to be used commercially for grain production
(EPA, 2009). 6
Cry proteins and vip3Aa20 in Bt maize kernels have been observed at varying levels
(Koziel et al., 1993; Mendelsohn, 1998, 1999) depending on the promoter gene present, which can either be cauliflower mosaic virus CaMV/35s promoter or maize phosphoenolpyruvate carboxylase (PEPC) promoter (Koziel et al., 1993). In the study of
Koziel et al. (1993), kernels of hybrids with CaMV/35s promoter were shown to have 10 times more Cry1Ab protein than the hybrids with PEPC promoter. Cry1Ab is regulated by
CaMV/35s promoter in transgenic maize Bt11 and MON810 events. Expression of Bt protein in both events was throughout the season in all plant parts including the grain and pollen (EPA, 2000; Sedlacek et al., 2001).
Coleopteran resistance genes
Corn rootworms (CRW) (Diabrotica spp.) are coleopteran field pests in the US responsible for the highest use of conventional insecticides such as organophosphates and pyrethroids. Corn rootworm larvae mainly feed on the roots, eventually causing lodging, hence the host’s water and nutrient absorption from the soil is greatly inhibited. Cry34Ab1 x Cry35Ab1 is a binary toxin co-expressed chiefly to control CRW and commercialized as a single event hybrid since 2006 or pyramided with either Cry3Bb1 or mCry3A (such as
Mir604 event) (Schnepf et al., 2005; Wang et al., 2017). Toxicity of Cry34Ab1 alone is limited and Cry35Ab1 alone has no toxicity at all; therefore, the binary form is necessary to achieve CRW larvae control since the specific binding of Cry35Ab1 is enhanced by
Cry34Ab1(Moellenbeck et al., 2001; Wang et al., 2017). Cry34Ab1 x Cry35Ab1 kills coleopteran larvae pests by binding to specific sites localized in the midgut lining of susceptible species. After successfully binding, gut paralysis and eventually death occurs 7 because of the disrupted ion flow in the midgut due to the formation of pores and bacterial sepsis. DAS59122-7 is an example of a maize event that has Cry34Ab1 x Cry35Ab1.
Diabrotica species reported to be effectively controlled include northern corn rootworm
(NCRW) (D. longicornis barberi Say), western corn rootworm (WCRW) (D. virgifera
LeConte) and Mexican corn rootworm (MCRW) (D. virgifera zeae LeConte). However, CRW resistance to these Cry3 proteins has been reported recently (Gassmann et al., 2014;
Wangila et al., 2015).
A modified cry3A gene from B. thuringiensis subsp. tenebrionis, i.e., mcry3A, is a constructed to optimize expression in maize. Mir604 event expressing mcry3A protein was registered in 2006 and is the third CRW-protected Bt maize in the market. It has unique biochemical properties beneficial for coleopteran resistance management in the field and has enhanced activity against the key corn rootworm species, WCRW and NCRW (EPA,
2006). All these proteins have no foreseeable human health and environmental risks.
Intestinal cells of mammals do not have the binding sites for delta- or beta-endotoxins of B. thuringiensis; therefore, humans, livestock, and non-coleopteran invertebrates are not affected. Moreover, Cry proteins easily degrade in the soil, which means they are unlikely to accumulate in the soil and have no potential to cause residue problems (EPA, 2010a;
EPA, 2010b).
Aspergillus flavus in maize
Aspergillus flavus Link is a ubiquitous saprophyte that causes diseases commonly in oil-rich crops such as maize, peanut, and cottonseed (Klich, 2007). A. flavus can survive and overwinter in plant residues as mycelium or sclerotia that would serve as the source of 8 new conidia for the succeeding cycle (Abbas et al., 2008). It was known for years as an asexual species bearing conidia and sclerotia; a sexual stage was reported in 2009 as
Petromyces flavus (Horn et al., 2009). Although morphologically diverse, A. flavus colonies largely produce yellow-green spores, and form sclerotia that are classified into two groups based on size: L strains (Group I) with sclerotia >400 mm in diameter and S strains (Group
II) with sclerotia <400 mm in diameter (Cotty, 1989; Horn, 2003). A. flavus is not considered an aggressive invader and does not necessarily reduce yield, but it may cause severe rotting of solanaceous crops and other fruits. In maize, it can cause discoloration and rotting of germs and whole kernels (Sauer et al., 1992). Since A. flavus is an opportunistic pathogen, higher incidence has been observed in hosts that are under stressed conditions and insect or mechanically damaged (Amaike & Keller, 2011). Healthy plant tissues are less prone to A. flavus extensive colonization but when subjected to heat stress or moisture deficit, reproductive structures of maize become readily susceptible to high levels of aflatoxins (O’Brian et al., 2007). A developing grain is also more susceptible to
A. flavus colonization if insect or mechanical damage is present (Diener et al., 1987). Other than invasion through wounds or damage, A. flavus is capable of entering maize through silks, kernels, and even produce aflatoxins in developing ears in an environment without any insect pressure (Jones et al., 1980).
Fungi attacking maize from production up to storage are often categorized as field or storage fungi. Field fungi colonize ripening grains in the field prior harvest. A. flavus is considered a storage fungus that can originate from the field but initiates invasion during storage, if conditions have a relative humidity of at least 78-84% and temperature between
30-35°C (Sanchis & Magan, 2004; Fleurat-Lessard, 2017). In a controlled environment 9 experiment conducted by Payne et al. (1985), 34°C day/30°C night temperature regime has led to strikingly higher A. flavus infection on maize kernels with 28% kernels infected after silk inoculation compared to a 26°C day/22°C conditions where only 2.4% of the kernels were infected. Storage fungi, in general, may increase the temperature and equilibrium moisture content (EMC) of the invaded grain at the level of relative humidity that favors their growth. Storage fungi may attack the embryo and endosperm of a kernel. The embryo is attacked by storage fungi at the lower limit of moisture content that permits fungal invasion (Sauer et al., 1992). At higher moisture contents, the endosperm is infected which significantly reduces caloric value of the grain. Other common storage fungi include
Curvularia spp. and Penicillium spp. (Williams & MacDonald 1983; Barney et al., 1995).
The major types of losses due to storage molds include quality reduction due to discoloration, heating and mustiness, various biochemical changes, mycotoxin contamination, and weight loss (Christensen & Kaufmann, 1969). Among these, the principal concern for A. flavus is mycotoxin contamination, particularly aflatoxins, the most potent mycotoxins associated with maize. Estimates of economic losses to aflatoxins are variable. According to Rubens & Cardwell (2003), economic losses due to aflatoxin contamination in maize were estimated to be approximately $2 million in Mississippi
(1998) and $15 million in Texas (1999). In a more recent estimate, market losses annually due to aflatoxin contamination were estimated as $163 million in the US. No animal health losses were reported for aflatoxin but animal mortality due to fumonisin consumption was
$0.27 million (Wu, 2006).
A. flavus is also the second-leading pathogen among the 20 other species of
Aspergilli that causes aspergillosis (Krishnan et al., 2009). Exposure to Aspergillus may also 10 cause aspergillosis which are often fatal in immunocompromised humans (Hedayati et al.,
2007). In North America, 65% of the reported childhood aspergillosis is caused by A. flavus
(Steinbach, 2005) and the in the year 2010, Tilak et al. identified A. flavus as the leading cause of mycotic keratitis, a fungal infection of the cornea, in India. A. flavus spores can be inhaled from contaminated grains and feeds. Frequent inhalation may lead in allergic reactions characterized by asthma, extrinsic alveolitis, or allergic bronchopulmonary aspergillosis. In animals, rabbits, domesticated chickens, geese, and turkey are quite susceptible to aspergillosis by A. flavus (Hedayati et al., 2007).
Aflatoxin contamination and toxicity
Aflatoxin is a secondary metabolite that was first associated with Aspergillus species in the year 1962, when thousands of poultry died in the United Kingdom due to Turkey X disease that resulted from feeding on aflatoxin-contaminated peanuts (Blout, 1961). Since then mycotoxin research revolutionized leading to screening of products for possible mycotoxin contamination. A. flavus is the most widely recognized aflatoxin producer, along with A. parasiticus and A. fumigatus; although there are also strains of A. flavus that are non- aflatoxin producers (Abbas et al., 2006). Maize and cottonseed are often contaminated with aflatoxin B1 (AFB1) produced by A. flavus (Klich, 1986). A. parasiticus, on the other hand, is more common in peanuts than any other crop; but, when A. flavus is also present in the infected peanut, A. flavus outcompetes A. parasiticus (Horn et al., 1995). A. flavus L strains produce aflatoxins B1 and B2 while S strains also produce G1 and G2. Production of aflatoxins by A. flavus is optimum at 28oC and 99% relative humidity (Sanchis & Magan,
2004), but the optimum temperature may vary between 25-30oC depending on the strain 11 and substrate (Klich, 2007). According to Sorenson et al. (1967), temperatures higher than
32oC strikingly hinder aflatoxin production despite enhanced proliferation of A. flavus in rice. However, in a recent study in maize grain, Medina et al., (2017) observed a significant increase in aflatoxin production when temperature was increased from 30 to 37oC based on the higher expression levels of aflatoxin biosynthesis genes.
Ingestion of high levels of aflatoxins in contaminated food or feed leads to aflatoxicosis. It has been a major problem in developing Asian and African countries (Lewis et al., 2005). Frequent intake of aflatoxins in low doses may result to chronic aflatoxicoses, impaired food consumption, and chances of liver cancer over time (Cardwell et al., 2004;
Farombi, 2006). Acute aflatoxicoses can occur by ingestion of high concentrations of aflatoxins in just one or few exposures. Consumption of aflatoxin contaminated maize have killed hundreds of people in Kenya in recent years where repeated outbreaks of acute aflatoxicosis occurred such as in year 1981-1982, 2001, 2004-2006, and 2008 (Ngindu et al., 1982; Lewis et al., 2005; Farombi, 2006; Probst et al., 2010). In 2005, consumption of maize contaminated with A. flavus S strain led to 150 human deaths (Strosnider et al.,
2006). In people infected with hepatitis B and C, commonly in East Asia and sub-Saharan
Africa, ingestion of aflatoxin raises by up to thirty-fold the risk of having hepatocellular carcinoma or liver cancer compared to exposure alone (Wu, 2006). Turner et al., (2003) reported that aflatoxin consumption is associated with immune system disorders and stunting in children. In the US, aflatoxicosis outbreaks were reported in 1998 and 2005-
2006 killing dogs that consumed commercial dry dog foods (Garland & Rigor, 2001;
Stenske et al., 2006). AFB1 is a potent natural carcinogen of the liver, kidney, lungs, and colon in humans and animals and the only mycotoxin categorized by the International 12
Agency for Research on Cancer (IARC) as a Class 1 carcinogen (WHO, 1993). AFB1 has caused liver tumors in many animals, and has been associated with immunotoxicity, reduced weight gain and productivity, and lower egg production in poultry (Bondy &
Pestkam 2000). In cattle, aflatoxicosis is manifested by reduced weight gain, liver and kidney damage, and reduced milk production (Keyl, 1978). Federal regulation of allowable amounts in food and feed have been established to prevent the risks associated with aflatoxin but allowable threshold levels vary among countries (Klich, 2007). The US Food and Drug Administration (FDA) determined an action level of 20 ppb in crops for human consumption, 100 ppb for breeding cattle, swine, and mature poultry; while European regulations are more stringent; for example, the allowable level is only 4 ppb of aflatoxins in peanuts for human consumption (Klich, 2007; Amaike & Keller, 2011).
Both colonization and mycotoxin production highly depend on prevailing storage conditions but the presence of A. flavus in maize does not necessarily guarantee the presence of aflatoxins (Plumlee & Galey, 1994). Also, optimum conditions for A. flavus growth and development are not always conducive for toxin production. A. flavus growth occurs at a broader range of conditions compared to aflatoxin production (Mannaa & Kim,
2017). According to Faraj et al. (1991), water activity (aw) influences aflatoxin production greater than temperature; but Mousa et al. (2013) found contradicting results. Medina et al.
(2017) concluded that variations in aw have a profound influence on the levels of aflatoxin production, as confirmed by more pronounced changes in aflatoxin-related gene expression.
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Mycotoxin reduction in Bt maize
Mycotoxin contamination in grain compromises grain quality and safety. Insect feeding predisposes maize kernels to infection and mycotoxin accumulation due to the open wounds they create. Insects also serve as vectors of fungal spores (Sinha, 1984;
Munkvold et al., 1999). Fumonisins, deoxynivalenol (DON), zearalenone, and aflatoxins are the four agriculturally important mycotoxins often associated with maize (Wu, 2007).
Fumonisins are produced mainly by Fusarium verticillioides and F. proliferatum and have been associated with esophageal cancer in humans, equine leukoencephalomalacia (ELEM) in horses, and pulmonary edema (PPE) in swine (Ross et al., 1992). DON, produced by F. graminearum and the related species F. culmorum, is considered as the most common mycotoxin in small grain cereals, especially wheat. It causes vomiting and nausea, immunosuppression, and loss of productivity in both humans and animals (Miller et al.,
2001). Zearalenone is also produced by F. graminearum and has been reported to cause estrogenic responses and vulvovaginitis in swine (Kurtz & Mirocha, 1978). Aflatoxins, on the other hand, are produced chiefly by A. flavus and A. parasiticus, and regarded as the most potent carcinogen of the liver in humans and causes liver and kidney damage in poultry and cattle among others (Keyl, 1978; Bondy & Pestkam, 2000; Cardwell et al.,
2004).
The presence of Bt proteins in maize deterring insect activity in the field consequently reduces infection of mycotoxigenic fungi and mycotoxin contamination in the grain. Mycotoxin reduction in Bt maize is greatly dependent on the species composition of insect pests present and the hybrid-environment interactions (Dowd, 2000). Bt maize can significantly lower mycotoxin contamination where insect damage is a major factor in 14 mycotoxin contamination (Wu, 2007). ECB, SWCB, and CEW infestation in the field have been shown to promote mycotoxin concentrations in maize (Dowd, 1988). In 1999, it was reported that hybrids expressing Cry1Ab had significantly reduced fumonisin accumulation compared to the near-isogenic controls based on a field study conducted in Iowa, USA
(Munkvold et al., 1999).
In the same study, it was observed that fumonisin B1 (FB1) concentration was significantly correlated with insect damage and Fusarium ear rot severity. In field studies in the US, when ECB or SWCB insect pressure is high, fumonisin levels in Bt maize were significantly reduced compared to the non-Bt isolines. FB1 concentrations in Bt maize events Bt11 and MON810 were significantly lower in naturally or manually infested ECB fields compared to their non-Bt near-isogenic counterparts. This significant reduction was only observed, however, on fields with ECB as the predominant pest and less significant with other pests such as CEW (Dowd, 1998; Munkvold et al., 1999; Munkvold & Hellmich,
2000; Dowd, 2001), due to incomplete control of CEW by Cry1Ab. Bowers et al., (2013) demonstrated that the hybrids expressing Cry1Ab or Cry1Ab stacked with Vip3Aa had significant reductions in ECB, CEW, and WBC damage, Fusarium ear rot severity, and fumonisin levels. In other parts of the world such as France, Italy, Turkey, Argentina, and
Philippines, field trials also showed lower fumonisin levels in Bt maize than the non-Bt isolines (Hammond et al., 2003; Bakan, et al., 2002; De la Campa et al., 2005). In a field study in Germany comparing three field plots of Bt maize, lower total fumonisin levels were observed in the Bt compared to the non-transgenic, but not DON levels (Papst et al., 2005).
Results of DON reduction in relation to Bt maize have been inconsistent because F. graminearum can successfully infect maize without insect damage. In field studies 15 conducted in Europe, relationship between ECB pressure and DON levels in Bt maize across years were inconsistent (Magg et al., 2002). But in years when ECB damage is high, significantly lower DON levels were observed in Bt maize than non-Bt, otherwise, no significant differences were observed (Schaafsma et al., 2002). In case of zearalenone, which is also produced by F. graminearum, lower levels of this mycotoxin were observed in
Bt maize in only certain field sites in France and Spain (Aulrich et al., 2001).
A. flavus infection and aflatoxin levels have long been correlated with insect damage
(Dowd, 1998), although like F. graminearum, A. flavus may infect maize without the aid of insects. Aflatoxins in maize were found to be less strongly correlated to insect damage compared to fumonisins. Field studies correlating insect damage, aflatoxin contamination, and Bt maize show mixed results likely because the insects controlled by Bt are not as important in predisposing maize to A. flavus infection as they are to F. verticillioides and F. graminearum (Miller, 1995; Wu, 2007). In the southern US where aflatoxins are a chronic problem, the most important Lepidopteran pests are CEW and FAW, which are not controlled adequately by the early Bt hybrids expressing only Cry1Ab. Aflatoxin reduction in Bt maize is highly influenced by the prevailing field conditions during A. flavus infection and the insect pest population present. In Southern US when aflatoxin levels in the field were high, aflatoxin contamination in Bt maize was significantly lower than the non-Bt isoline. However, during the years where aflatoxin levels were low in both Bt and non-Bt maize, significant reduction in the Bt maize was not observed (Wiatrak et al., 2005). In a study conducted by Buntin et al. (2001), although significant reduction of insect damage was observed in Bt11 and MON810 hybrids compared to the non-Bt isolines, aflatoxin levels were comparable between Bt11, MON810, and the non-Bt hybrids. Similarly, Odvody 16 et al. (2000) found significantly reduced insect damage in Bt maize in Texas fields compared to non-Bt but results of aflatoxin levels were comparable and inconsistent. In a three-year study in Mississippi, only in one year did they observed lower aflatoxin levels in
Bt maize than non-Bt isolines (Bruns & Abbas, 2006). In terms of Bt maize impact on severity of Aspergillus ear rot, Maupin et al. (2001) found that the differences were insignificant. Meanwhile, Headrick (2006) found that in Bt events with protection against
CEW and FAW, which are insects closely associated to aflatoxin contamination in maize, aflatoxin reductions were consistently observed in Bt maize compared to non-Bt. The same trend was observed by Abbas et al. (2008), where fungal propagules and aflatoxin levels were both reduced in Bt compared to the non-Bt isogenic line. There are variety of factors that may affect the influence of Bt in mycotoxin accumulation. Nonetheless, the benefit from using Bt maize to lower fumonisin and aflatoxin contamination annually was estimated to be around $8.8 M and $14.1 M, respectively (Wu et al., 2006).
Most recent studies in Bt maize-mycotoxin relationship are in the field and has not been examined in post-harvest conditions. Insect activity and feeding in storage causes grain temperature and MC of grain to rise, predisposing kernels to colonization by mycotoxigenic fungi. It was shown that aflatoxin levels were higher in maize grain infested with maize weevils and A. flavus than in grains with A. flavus only (Beti et al., 1995). Bt proteins such as Cry1Ab, mCry3A, Cry1Fa2 in Bt11, Mir604 and TC1507 events, respectively, are also expressed in the grain and have around 1-2 ng/mg mean expression levels in the grain by dry weight (Table 2) (EPA, 2010b, 2007). MacKenzie et al. (2007) found that Bt proteins can still be present in ground Bt maize after 90 days. The presence of
Bt proteins in the grain suggests the possibility of insect protection during grain storage. 17
Among the few studies conducted under post-harvest conditions showed Bt maize was effective against stored-grain insects such as Indianmeal moth and angoumois grain moth
(Sitotroga cerealella Olivier) (Giles et al., 2000; Sedlacek et al., 2001; Hanley et al., 2004).
According to Munkvold et al. (1999), if insecticidal proteins are expressed in all plant tissues of a Bt hybrid, then it is likely to have more indirect mycotoxin reductions as a result of greater efficiency in reducing insect activity.
Indianmeal moth biology, ecology, and damage
A stored-grain insect pest native to South America, Indianmeal moth (IMM) (Plodia interpunctella Hübner), is a Lepidopteran pyralid moth that is found almost everywhere in all continents except Antarctica (Rees, 2004; Mohandass et al., 2007). Colonies of IMM can be found in household bins, storage facilities, ocean freight shipments, and imported food products (Cox & Collins, 2002). Unlike other pyralid moth species, IMM oviposition is mainly influenced and oriented towards food odor (Deseo, 1976; Phillips & Strand, 1994) and not much affected by diurnal cycles and circadian rhythms (Bell, 1981). Eggs are laid on the surface, singly or typically spatially aggregated, with one adult female capable of laying 60-400 eggs that hatch within 2 to 14 days (Mullen & Arbogast, 1977; Arbogast &
Mullen, 1988). The larval phase is the only stage that causes damage by feeding externally on grain. Young larvae are white with brown head and turn yellowish-white as they grow older, reaching at most 12 mm in length. Larvae pupate in silk cocoons from which adults emerge after 7 days under optimal conditions. The entire life cycle of IMM may take between 30-300 days; at least 28 days under optimum conditions of 30-35°C and 25% relative humidity. 18
The duration of life cycle is highly variable because developmental times depend greatly on the kind of food source and environmental conditions particularly differences in protein and moisture levels, respectively (Giles et al., 2000); therefore, it has been challenging to define stage-specific developmental times. A period of suspended development, or diapause, occurs after feeding ceases in the last or 5th instar. Diapause may also happen early or be induced by low temperature or photoperiod of less than 13 hours
(Tzanakakis, 1959; Bell, 1976). In storage facilities where temperatures are uncontrolled, larvae may undergo diapause during colder months and abruptly increase in population in early spring (Mason, 2003). Field studies in the US have shown that IMM only has 1-3 generations peaking during the summer months (Arbogast et al., 2000; Campbell et al.,
2002; Nansen et al., 2004). IMM may migrate from outside going inside a storage facility at the beginning of a season (Doud & Phillips, 2000; Campbell & Mullen, 2004).
In the study of Giles et al. (2000), adult survivorship of IMM on cracked stored maize after 60 days ranged from 36.5-73%. Similarly, Kaliyan et al. (2005) observed that a higher percentage of broken kernels resulted in higher larval survival from 6.7% only in containers without broken kernels to 63.8% survival in containers with 7% broken kernels of yellow dent corn. Although mortality is associated with the inability of IMM larvae to penetrate intact kernels, still a fraction of the population that survives can cause economic losses in storage
(Hockensmith et al., 1986; Mbata, 1990). Aside from direct feeding of the larvae, silken thread formed by the larvae binds grain, larval frass, and cast skin together giving off an unpleasant odor (Mohandass et al., 2007). This contributes to loss due to quality reduction aside from the impact of feeding damage to the infested grain. Infestation of IMM can also 19 cause indirect economic costs through pest control inputs and consumer complaints
(Phillips et al., 2000).
IMM is susceptible to Cry proteins, such as Cry1Ab and Cry1Ac (Johnson et al.,
1998). In the study of Giles et al. (2000), Cry9C and Cry1Ab expressed by CaMV/35s promoter decreased survivorship, developmental time, and adult body length of IMM, and showed an effect up to 4 or 5 months after harvest. Likewise, Cry1Ab expressed in N6800Bt
(Bt11 event) and P33V08 (MON 810 event) resulted in longer developmental time (33-43 days), reduced fecundity (58-67%) and emergence (7.4-35.4%) of IMM. Emergence of angoumois grain moth was also significantly lower (19.8-22.8%). These effects on IMM were not observed in grain of a hybrid with E-176 event, which expresses Cry1Ab proteins, but not in kernels (Sedlacek et al., 2001). There is also a biological insecticide, called DiPel, that has a cry profile of Cry1Aa/Cry1Ab/Cry1Ac/Cry2A registered for use against IMM and almond moth (Cadra cautella Walker) on stored grain.
There are reports of resistance development to insecticides and commercial Bt products among IMM populations (Summer et al., 1988, McGaughey & Johnson 1992,
Tabashnik & McGaughey, 1994). This was first observed in grains collected from the
Midwest (McGaughey, 1985). This resistance development could reduce the benefit of Bt maize hybrids against IMM (McGaughey, 1994; McGaughey & Johnson, 1994; Gould, 1998;
Herrero et al., 2001). However, all reports of resistance to Bt in IMM were from laboratory populations and there is no direct evidence yet of field resistance (Mohandass et al., 2007).
20
Maize weevil biology, ecology, and damage
The maize weevil (MW) (Sitophilus zeamais Motschulsky), also known in the US as the greater grain weevil, is a coleopteran (Curculionidae) insect pest of several stored grains, including maize, sorghum, rice, and wheat. It can be found abundantly in warm and tropical areas worldwide and is considered a major pest of maize in the US and Sub-
Saharan Africa (Smale et al., 2011). MW cannot overwinter in temperate areas unless in heated warehouses. This beetle has a small snout, fully developed wings but seldom flies, and has four reddish markings on the wing covers. Overall appearance is similar to the rice weevil (S. oryzae Linnaeus), but MW is much larger and darker.
Maize weevil adults oviposit throughout most of their adult life, in stored cereal grains or in the field at temperatures between 15-35°C, optimum around 25°C. It attacks in the field when the moisture content of the grain has fallen to 18-20% (Parugrug & Roxas,
2008). An adult female can lay up to 150 eggs; 50% of which are laid during the first 4-5 weeks. Unlike IMM, MW is an internal feeder; the eggs are laid within the grain and hatch after few days or about 6 days at 25°C into a soft, white, legless grub that starts feeding on the interior of the maize kernel (Howe, 1972). It usually takes a minimum of 30 days to complete its life cycle from egg to larva with 4 instars and pupal stages and all these developmental phases occur within the chambers bored in the grain; thus, the infestation is often unseen (Longstaff, 1981; Jacobs & Calvin, 1988). Duration of life cycle depends on the type, quality, and quantity of the infested grain, in different maize varieties, and average development period of maize weevil vary from 31-37 days at 27°C and 70% RH (Danho et al., 2002; Suleiman et al., 2015). 21
Two stages of MW actively feed on the grain, one during the adult stage where adults chew cavities to oviposit eggs and second is during the larval stage. The adult then seals the cavity to protect the egg by creating an egg-plug, a waxy secretion produced by the female adult (Howe, 1972). Upon hatching, the grub excavates a tunnel through feeding beginning from the inside of the grain until pupation commences. The newly developed adult emerges from pupa chewing its way out of the kernel leaving a definite circular hole.
This hole, large enough for an adult weevil, is a characteristic of damage by MW. Adults are long-lived, several months to one year, and are capable of consuming kernels completely during storage producing a considerable amount of grain dust. This reduces the bulk density, moisture content, and starch value of the grain. Moreover, the feeding damage inflicted predisposes grain to mold development (CGC, 2013; Muzemu et al., 2013;
Suleiman et al., 2015).
Attack of MW starts in the field, but damage is more serious during storage, reaching up to 20-90% weight loss in untreated maize in the tropics or in susceptible varieties such as dent and sweet corn (Fikremariam et al., 2009; Muzemu et al., 2013). Indirect economic impact of MW damage also includes lower nutritional and market value of the grain (Keba
& Sori, 2013). There are weevil resistant varieties of orange flint corn, yellow and white popcorn that can be used to minimize problems (Suleiman et al., 2015). Several recent studies focus on screening and evaluating maize varietal resistance to MW (Fikremariam et al., 2009; Muzemu et al., 2013; Suleiman et al., 2015). Lepidopteran resistance genes
Cry1Ab/Cry1b were shown to have no influence on MW based on several life cycle parameters (Pittendrigh et al., 1997; Hansen et al., 2013). Vip1/2 binary toxin reported to work against coleopterans resulted in 60% mortality of maize weevil in laboratory assays 22 using Vip1/2 extracts from a local strain of B. thuringiensis (Shingote et al., 2013).
Transgenic maize studies only involved avidin that caused MW mortality significantly higher than the non-transgenic maize (21 ± 2% vs. 5 ± 1%, respectively, p< 0.01) (Kramer et al., 2000) and a similar effect was observed in granary weevil (S. granarius Linnaeus) infesting avidin transgenic wheat reaching 100% mortality in transgenic plants after 21 days (Abuoseadaa et al., 2015).
Grain storage ecosystem and management
The concept of the stored-grain ecosystem explains interrelations between abiotic and biotic factors that can be taken into consideration to ultimately manage stored grain losses to a minimum (Sinha, 1995). Abiotic factors include temperature, aw, gas composition, and the nature of substrate (Miller, 1995; Magan & Olsen, 2004); which have an additive effect on insect survival and progeny (Mason & Strait, 1997). The major biotic factor is the stored grain itself, then the insects, and microorganisms (Calderon, 1981).
These factors influence the dominance of fungi, more importantly the mycotoxigenic species, that impact quality and nutritive components of the grain (Sanchis & Magan, 2004;
Chulze, 2010). Fungal spores in the grain ecosystem stay dormant until the conditions became suitable for development, mainly in response to temperature and relative humidity that drives moisture in and out of the grain until an equilibrium moisture content (EMC) is attained (Loewer et al., 1994).
The storage structure is also an important abiotic factor to consider (Sanchis &
Magan, 2004). A good storage structure should satisfy the following parameters: grains are kept dry; maintained at a uniform temperature; protected from insect attack, and; rodents 23 and birds are completely excluded. In developed countries, maize is mostly stored in commercial infrastructures with good control of abiotic factors to control pests. In Sub-
Saharan Africa, the common practice is to sun-dry maize by laying cobs on the ground for
5-7 days and then transfer into traditional granaries for storage such as open-air cribs made of woven bamboo/twigs, mud silos, or a raised platform made up of tree poles and thatch grasses (Markham et al., 1994; Kankolongo et al., 2008). These systems are often inadequate in providing a good dry environment for maize especially in humid and semi- humid zones (Fandohan et al., 2003). Grain can also be stored in bags such as woven polypropylene (PP) that are widely available and used. PP bags are affordable but the use of fumigants along with the bags increases the cost. In recent years, the use of hermetic storage devices is becoming common in Asia and Africa (De Groote et al., 2013; Njoroge et al., 2014). Several studies have shown the benefits of hermetic storage through reduced aflatoxin accumulation, MW and larger grain borer (Prostephanus truncates Horn) infestations, and grain weight loss (Njoroge et al., 2014; Walker et al., 2018).
A. flavus association with insects
Insects, in general, can promote fungal infection process by providing points of entry for invasion and carrying fungal propagules. This role of insects to fungal infection depends on the present insect population, resistance of plant to the fungus, and the conditions that may favor fungal growth and development. Aspergillus symptoms and aflatoxin production in the field have been related to activities of ear-feeding insects such as ECB, CEW, WBC, SWCB, and FAW; where increases of aflatoxin contamination were observed in infested pre-harvest maize (Christensen & Schneider, 1950; Widstrom et al., 24
1975; Fennell et al., 1975; La Prade et al., 1977; Setamou et al., 1998; Buntin, 2008). Visual damage on kernels by these insects may indicate possible aflatoxin contamination (Fennell et al., 1977). This association in maize was recognized first by Riley (1882) when he reported that molds appear on tips of maize soon after insect larvae infestation occurred. It was then observed that high severity and incidence of moldy ears happen during years where insect infestations were high (Garman & Jewett, 1914). Windham et al. (1999) observed that commercial hybrids infested with SWC and inoculated with A. flavus had significantly higher aflatoxin levels than in hybrids inoculated with A. flavus alone.
However, in some years, SWC infestation didn’t result to increased aflatoxin contamination.
Like any other insect-pathogen relationship, Aspergillus infection, aflatoxin contamination, and insect damage in the field are heavily influenced by environmental conditions. A. flavus geographical distribution has been found to be relatively uniform, but aflatoxin levels showed distinct regional variation because of differences in environmental conditions
(Fennell et al., 1977).
A. flavus is known to produce aflatoxins that are hazardous to human and animal health, however, its role as an insect pathogen remains obscure (Dowd, 2003). It was shown that A. flavus was not specialized to a certain host based on the several strains isolated from different hosts, i.e., from plant, insects, animals, and even humans. Aside from aflatoxins, A. flavus produces enzymes, such as proteases, that can potentially degrade the protective exoskeleton of insects (St. Leger et al., 2000). In storage, since there are mycotoxigenic fungi such as Fusarium spp. and Aspergillus spp. colonizing maize, it is advantageous for insects to be resistant to toxic metabolites produced by these fungi.
According to Dowd (2003), insects feeding on moldy kernels are highly resistant to 25 mycotoxins, and some insects even benefit from the presence of aflatoxin-producing fungi.
Caterpillar ear borer (Mussidia nigrivenella Ragonot) is among the insects not affected by A. flavus even at high aflatoxin levels. In Africa, A. flavus was consistently found associated with caterpillar ear borer and in a two-year survey, presence of this insect was significantly correlated with aflatoxin (r=0.36 and 0.52) (Setamou et al., 1998).
Mechanisms of Aspergillus-insect interactions
Interaction is described by Mills (1982) as two populations occurring together that have definite influence on the other, unlike the term association, which only involves coexistence but without affecting each other. Interactions happening in storage mainly depend on the type of grain and levels of moisture influenced by the presence of insects or microorganisms. Interactions may be beneficial or detrimental for each or both insect and pathogen or may involve fulfilling specific roles in terms of establishment in maize; in the context of A. flavus, some of these interactions are briefly discussed below:
As vectors. Traditionally, interactions between insects and fungi are viewed in the perspective of insect transmission of inoculum and the biotic components of stored grain
(Mills, 1982). Being vectors of fungal propagules is a major role of insects, and this happens simultaneously with the establishment of entry points for fungi through the feeding sites
(Fennell et al., 1975; Fennell et al., 1978). This largely explains the long-standing positive correlation of mycotoxin contamination with insect populations (Lillehoj et al., 1978; Sobek
& Munkvold et al., 1999; Bowers et al., 2013). Insects may disseminate spores in the field or within the storage bins or containers, and some may be capable of transporting propagules 26 over long distances. However, no stored-grain insect found so far has a mycangium or specialized structure adapted to carry spores over long distances (Dunkel, 1988). La Prade
& Manwiller (1977) surmised that maize weevil movement within the container disseminates the spores but they are non- or inefficient vectors of A. flavus over long distances. Likewise, Dix et al. (1984) observed that patterns of A. flavus sporulation in test bins coincided with the pattern of movement of maize weevil. In a recent study by Ferreiro-
Castro et al. (2012), it was shown that the MW carried spores of A. flavus from inoculated grain to uninoculated grain and higher aflatoxin contamination was detected in grain with both A. flavus and MW compared to without the maize weevil. Presence of A. flavus internally and externally in or on insects was also noted by Lillehoj et al. (1975), where
1.0% external occurrence of A. flavus on test larvae was similar to the 1.7 to 3.1% occurrence of the fungus in maize insects observed in an earlier study of Fennell et al.
(1977).
Providing entry points. Insect feeding sites make the host more vulnerable to fungal attack and further colonization. Insect frass left in the feeding sites may serve as media for fungal growth and sporulation. A. flavus, as many pathogens, may enter host tissues through insect injury. Christensen and Schneider (1950) demonstrated that the ECB provided an avenue of entrance for Aspergillus spp. among other fungi. MW was also noted to enhance mold proliferation likely during its oviposition, and larval or metabolic activities (Sinha, 1984; Dunkel, 1988). This explains why fungal incidence was found to be highest inside a tunnel bored by an insect (Baba-Moussa, 1998, as cited by Schultess,
2001). 27
Attraction/sustenance/repellence. The metabolites produced by fungi and from the biochemical changes in maize attracts insects into host or induces insect movement, feeding, and sexual activity (Cox & Collins, 2002; Franco et al., 2017). Insects that are attracted to moldy maize kernels are likely not harmed by the fungi’s presence and instead favor the insect’s development (Dunkel, 1988). However, more studies should be done on how and when these volatiles trigger insect movement, succession, and fungal infection in the stored-grain ecosystem (Arbogast & Mullen, 1988; Jian & Jayas 2012). Van Wyk et al.
(1959) demonstrated that a flour inoculated with a mixture of Aspergillus spp. spores have four times more beetle present compared to the non-inoculated. In some cases, stored- grain insects such as larger black flour beetle (Tribolium destructor Uyttenboogart) may even voraciously feed on spores of Cladosporium, Fusarium moniliforme and Scopulariopsis brevicaulis (Sinha, 1971). Some can either feed solely on molds or on grain to complete their life cycle, e.g., red flour beetle (Tribolium castaneum Herbst), while some only feed on the grain and not the molds, e.g., maize weevil (Ferreira-Castro et al., 2012). Insects that are not associated with Aspergillus might be repelled by the volatiles produced by it. Sinha
(1971) demonstrated that pure cultures of Aspergillus spp. repel most stored grain insects except the foreign grain beetle (Ahasverus advena Waltl).
Creating hot spots. During storage, there is transformation on stored-grain ecosystems due to metabolic activities of insects resulting in increased relative humidity, temperature, and moisture content of grains (Sinha, 1971; Hambleton, 1979; Lacey et al.,
1980), which promotes germination and development of postharvest fungi in the intergranular spaces (Lacey et al., 1980). Subsequently, not only the insects but also the molds contribute to this increase in temperature or heating which will continue even after 28 insects are killed (Sauer et al., 1992). Moreover, the presence of volatiles, body fragments of insects, and fungal metabolites simultaneously contributes to these changes (Sone,
2001). Both insects and fungi can initiate hot spots in storage; hot spots initiated by insects are typically infested by fungi because heat and water produced by insects benefit the development of fungi. Fungi proliferate around or inside the hot spot and induce temperature increase up to 65oC (Sinha & Wallace, 1965). Dix (1984) showed that in grain storage, Penicillium spp. often precedes A. flavus infection and the presence of MW enhanced Penicillium spp. growth due to an increase in metabolic water production. This in turn also favored the growth A. flavus. Similarly, Barney et al. (1995) observed that
Penicillium spp. and A. glaucus were both significantly influenced by the presence of maize weevil.
Inhibition of growth. Instead of promoting the development of insect populations in maize, pathogens can also be detrimental to the insects occupying the same niche. A. flavus produces degradative enzymes such as proteases (St. Leger et al., 2000) and lipases
(Long et al., 1988) that can potentially degrade the exoskeleton of insects (Dowd, 2003). A. flavus and A. parasiticus may produce aflatoxin and kojic acid that were shown to be insecticidal (McMillian et al., 1980). In the study of Matsumura & Knight (1967), aflatoxin resulted in 90% mortality of adult fruit fly (Drosophila melanogaster Meigen) and 71% in house fly (Musca domestica Linnaeus). MW was also found to be adversely affected by aflatoxicosis caused by A. flavus, but most adults were still able to survive and disseminate a high density of spores (Dix, 1984). Many reports described aflatoxin toxicity to insects but there are also that can tolerate exposure to aflatoxins. Navel orangeworm (Amyelois transitella Walker) is remarkably resistant to aflatoxin (Niu et al., 2009), likely due to its 29 exceptionally active detoxification system that is capable of converting aflatoxins to less harmful metabolites (Lee & Campbell, 2000). Similarly, foreign grain beetle was found to be less sensitive to aflatoxin by 200-2000 times more than the other stored-grain insects
(Zhao et al., 2018).
In certain cases, it is the fungal growth that is inhibited by the insect. Yeh (1979) showed that in the presence of confused flour beetle (Tribolium confusum du Val), no A. flavus incidence in the bins containing maize was observed, but A. flavus incidence is 3.3% if the bin has foreign grain beetle; probably due to the quinones secreted by the confused flour beetle (Van Wyk et al., 1959).
Overview and Objectives
Most A. flavus and aflatoxin studies related to insect interactions have focused on injury occurring in the field (Fennell et al., 1975; Lillehoj et al., 1975; Widstrom et al. 1975;
La Prade et al., 1977; Setamou et al., 1988). The coexistence of insect pests and fungal pathogens in maize field often results in higher infestation, disease severity, or mycotoxin contamination (Munkvold et al., 1999; Munkvold, 2003; Mouhoube, 2002; Dowd, 2003;
Bowers et al., 2013). This relationship grants that insect control is undeniably an important component in managing diseases and a key strategy in mitigating mycotoxin risks in maize.
At present, Bt maize provides the most effective way to suppress insect pest populations and has been proven to indirectly reduce mycotoxin levels in the field. Bt proteins are also expressed in the grain (Table 1); this warrants a study on their potential impact on stored- grain insects and aflatoxin contamination in storage. 30
The general objective of this study was to assess the interactions between an aflatoxin producing strain of A. flavus and the lepidopteran and coleopteran stored-grain insects, Indianmeal moth and maize weevil, respectively, in transgenic maize under controlled storage conditions. Specific objectives were as follows: first, to assess the effects of Indianmeal moth or maize weevil infestation on A. flavus growth and vice versa; second, to measure the impacts on grain quality in terms of aflatoxin contamination, damage, and grain weight loss; and lastly, to compare insect-fungus interactions in conventional and Bt maize hybrids with lepidopteran and coleopteran resistance genes.
Literature Cited
Abbas, H.K., Cartwright, R.D., Xie, W., Mirocha, C.J., Richard, J.L., Dvorak, T.J., Sciumbato, G.L. and, Shier, W.T. 1999. Mycotoxin production by Fusarium proliferatum isolates from rice with Fusarium sheath rot disease. Mycopathologia 147(2): 97-104. doi:10.1023/A:1007147813326.
Abbas, H.K., Zablotowicz, R.M., Bruns, H.A., and Abel, C.A. 2006. Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates. Biocontrol Science and Technology 16:437-449.
Abouseadaa, H.A., Osman, G.H., Ramadan, A.M., Hassanein, S.E., Abdelsattar, M.T., Morsy, Y.B., Alameldin, H.F., El-Ghareeb, D.K., Nour-Eldin,H.A., Salem, R., Gad, A.A., Elkhodary, S.E., Shehata, M.M., Mahfouz, H.M., Eissa, H.F., and Bahieldin, A. 2015. Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects. BMC Plant Biology 15: 183.
Amaike, S., and Keller, N.P. 2011. Aspergillus flavus. Annual Review of Phytopathology 49(1): 107-133. doi: 10.1146/annurev-phyto-072910-09522.
Arbogast, R.T and Mullen, M.A. 1988. Insect succession in a stored-corn ecosystem in southeast Georgia. Annals of Entomological Society of America 81:899-912.
Arbogast, R.T. and Mullen, M.A. 1978. Spatial distribution of eggs by ovipositing Indianmeal moths, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae). Researches on Population Ecology 19:148–154. Available from: http://www.argenbio.org/adc/ uploads/15_years_Executive_ summary_of_GM_crop_in_Argentina.pdf 31
Aulrich, K., Bohme, H., Daenicke, R., Halle, I., and Flachowsky G. 2001. Genetically modified feeds (GMO) in animal nutrition: Bacillus thuringiensis (Bt) corn in poultry, pig and ruminant nutrition. Archives of Animal Nutrition 54:183–95.
Bakan, B., Melcion, D., Richard-Molard, D., and Cahagnier, B. 2002. Fungal growth and Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain. Journal of Agricultural and Food Chemistry 50(4): 728-31.
Barney, R.J., Price, B.D., Sedlacek, J. D., and Siddiqui, M. 1995. Fungal species composition and abundance on stored corn as influenced by several management practices and maize weevil. (Coleoptera: Curculionidae). Crop Protection 14(2): 159-164. doi: 10.1016/0261 2194(95)92871-J.
Beck, D.L. 2002. Management of hybrid maize seed production. Mexico, D.F.: CIMMYT.
Bell, C.H. 1976. Factors governing the induction of diapause in Ephestia elutella and Plodia interpunctella. Physiological Entomology 1: 83–92.
Bell, C.H. 1981. The influence of light cycle and circadian rhythm on oviposition in five pyralid moth pests of stored products. Physiological Entomology 6: 231–239.
Blout, W.P. 1961. Turkey “X” disease. Turkeys. 52: 55–58.
Bondy, G.S. and Pestka, J.J., 2000. Immunomodulation by fungal toxins. Journal of Toxicology and Environmental Health 3: 109-143.
Bowers, E. Hellmich, R., and Munkvold, G. 2013. Vip3Aa and Cry1Ab proteins in maize reduce Fusarium ear rot and fumonisins by deterring kernel injury from multiple Lepidopteran pests. World Mycotoxin Journal 6(2): 127-135.
Bowers, E., Hellmich, R. and Munkvold G. 2014. Comparison of Fumonisin Contamination Using HPLC and ELISA Methods in Bt and Near-Isogenic Maize Hybrids Infested with European Corn Borer or Western Bean Cutworm. Journal of Agriculture Food and Chemistry 62(27): 6463-6472 dx.doi.org/10.1021/jf5011897.
Bruns, H.A., and Abbas, H.K. 2006. Planting date effects on Bt and non-Bt corn in the mid- South USA. Agronomy Journal 98:100–6.
Buntin, G.D. 2008. Corn expressing Cry1Ab or Cry1F endotoxin for fall armyworm and corn earworm (Lepidoptera: Noctuidae) management in field corn for grain production. Florida Entomologist 91(4):523–30 dx.doi.org/10.1653/0015-4040-91.4.523. 32
Buntin, G.D., Lee, R.D., Wilson, D.M., and McPherson, R.M. 2001. Evaluation of YieldGard transgenic resistance for control of fall armyworm and corn earworm (Lepidoptera: Noctuidae) on corn. Florida Entomologist 84:37–42.
Burkness, E.C., Dively, G., Patton, T., Morey, A.C., and Hutchison, W.D. 2010. Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy againts Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions: Implications for resistance management. GM Crops. 1(5): 337-343. doi: 10.4161/gmcr.1.5.14765.
Campbell, J.F. and Mullen, M.A. 2004. Distribution and dispersal behavior of Trogoderma variabile and Plodia interpunctella outside a food processing plant. Journal of Economic Entomology 97(4):1455-64.
Campbell, J.F., Mullen, M.A., and Dowdy, A.K. 2002. Monitoring stored product pests in food processing plants with pheromone trapping, contour mapping and mark recapture. Journal of Economic Entomology 95: 1089–1101.
Canadian Grain Commission. 2013. Maize weevil Sitophilus zeamais Motschulsky [WWW Document]. URL https://www.grainscanada.gc.ca/storage-entrepose/pip-irp/mw- cr-eng.htm
Cardwell, K.F., and Henry, S.H. 2004. Risk of exposure to mitigation of effects of aflatoxin on human health: a west African example. Journal of Toxicology 23:217-47.
Chakraverty, A., Mujumdar, A.S., and Ramaswamy, H.S. 2003. Handbook of postharvest technology: cereals, fruits, vegetables, tea, and spices. 1st ed. Vol. 93: CRC Press. 912p.
Christensen, C., and Kaufman, H. 1969. Mycotoxins and grain quality. Grain storage: the role of fungi in quality loss. University of Minnesota, Press Minneapolis, 76-93pp.
Christensen, J.J., and Schneider, C.L. 1950. European corn borer (Pyrausta nubilalis Hbn.) in relation to shank, stalk, and ear rots of corn. Phytopathology 40: 284-291.
Chulze, S. 2010. Strategies to reduce mycotoxin levels in maize during storage: a review. Food Additives and Contaminants 27(5): 651-657.
Cotty, P.J. 1989. Virulence and cultural characteristic of two Aspergillus flavus strains pathogenic on cotton. Phytopathology 79:808–14.
Cox, P.D., and Collins, L.E. 2002. Factors affecting the behaviour of beetle pests in stored grain, with particular reference to the development of lures. Journal of Stored Product Research 38:95–115. 33
Danho, M., Gaspar, C., and Haubruge, E. 2002. The impact of grain quantity on the biology of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae): oviposition, distribution of eggs, adult emergence, body weight and sex ratio. Journal of Stored Products Research 38(3): 259-266. doi:10.1016/S0022-474X(01)00027-3.
De Groote, H., Kimenju, S.C., Likhayo, P., Kanampiu, F., Tefera, T., and Hellin, J., 2013. Effectiveness of hermetic systems in controlling maize storage pests in Kenya. Journal of Stored Product Research 53: 27-36.
De la Campa, R., Hooker, D.C., Miller, J.D., Schaafsma, A.W., and Hammond BG. 2005. Modeling effects of environment, insect damage, and Bt genotypes on fumonisin accumulation in maize in Argentina and the Philippines. Mycopathologia 159:539- 52.
Deseo, K.V. 1976. The oviposition of the Indian meal moth (Plodia interpunctella Hubn., Lep., Phycticidae) influenced by olfactory stimuli and antennectomy. Symposium of the Biological Society of Hungary 16: 61–65.
Diener, U.L., Cole, R.J., Sanders, T.H., Payne, G.A., Lee, L.S., and Klich, M.A. 1987. Epidemiology of aflatoxin formation by Aspergillus flavus. Annual Reviews in Phytopathology. 25: 249–270.
Dix, D.E. 1984. Interactive Bionomics of the Maize Weevil, Sitophilus zeamais Motschulsky, and Aspergillus flavus Link. University of Georgia, Athens, GA, 129 pp.
Doud, C.W., and Phillips, T.W. 2000. Activity of Plodia interpunctella (Lepidoptera: Pyralidae) in and around flour mills. Journal of Economic Entomology 93: 1842– 1847.
Dowd, P.F. 1998. The involvement of arthropods in the establishment of mycotoxigenic fungi under field conditions. In Mycotoxins in Agriculture and Food Safety, ed. KK Sinha, D Bhatnagar, pp. 307–50. New York: Marcel Dekker.
Dowd, P.F. 2000. Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: Utility and limitations. Journal of Economic Entomology 93: 1669-1679.
Dowd, P.F. 2001. Biotic and abiotic factors limiting efficacy of Bt corn in indirectly reducing mycotoxin levels in commercial fields. Journal of Economic Entomology 94(5):1067- 74.
Dowd, P.F. 2003. Insect management to facilitate preharvest mycotoxin management. Journal of Toxicology 22(2): 327-350. 34
Dunkel, F.V. 1988. The relationship of insects to the deterioration of stored grains by fungi. International Journal of Food Microbiology 7: 227–244.
Eichenseer, H., Strohbehn, R., and Burks, J. 2008. Frequency and severity of Western bean cutworm (Lepidoptera: Noctuidae) ear damage in transgenic corn hybrids expressing different Bacillus thuringiensis cry toxins. Journal of Economic Entomology 101: 555-563.
Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., and Koziel, M.G. 1996. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proceedings of the National Academy of Sciences of the United States of America 93: 5389-5394.
Fandohan, P., Hell, K., Marasas, W.F.O., and Wingfield, M.J. 2003. Infection of maize by Fusarium species and contamination with fumonisin in Africa. African Journal of Biotechnology 2(12): 570-579.
Faraj, M.K., Smith, J.E., and Harran, G. 1991. Interaction of water activity and temperature on aflatoxin production by Aspergillus flavus and A. parasiticus in irradiated maize seeds. Food Additive and Contaminants 8:731-736.
Farombi, E.O. 2006. Aflatoxin contamination of foods in developing countries: implications for hepatocellular carcinoma and chemopreventive strategies. African Journal of Biotechnology 5:1–14.
Fennell, D. I., Kwolek, W.F., Lillehoj, E.B., Adams, G.L., Bothast, R.J., Zuber, M.S., Cahert, O. H.W., Guthrie, D., Bockholt, A.J., Manwiller, A., and Jellum, M. D. 1977. Aspergillus flavus presence in silks and insects from developing and mature corn ears. Cereal Chemistry 54:770-778.
Fennell, D.I., Lillehoj, E.B. and Kwolek, W.F. 1975. Aspergillus flavus and other fungi associated with insect-damaged field corn. Cereal Chemistry 52:314-321.
Fennell, D.I., Lillehoj, E.B., Kwolek, W.F., Guthrie, H.W., Sheeley, R., Sparks, A.N., Widstrom, N.W., and Adams G.L. 1978. Insect larval activity on developing corn ears and subsequent aflatoxin contamination of seed. Journal of Economic Entomology 71: 624-628.
Fikremariam, A., Tadele, T., Stephen, M., Yoseph, B., and Stefan, V., 2009. Resistance of maize varieties to the maize weevil Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae). African Journal of Biotechnology 8: 5937-5943.
Fleurat-Lessard F. 2017. Integrated management of the risks of stored grain spoilage by seedborne fungi and contamination by storage mould mycotoxins: an update. Journal of Stored Products Research 71: 22–40. 35
Franco, F.P., Moura, D.S., Vivanco, J.M., and Silva-Filho., M.C. 2017. Plant–insect–pathogen interactions: a naturally complex ménage à trois. Current Opinion in Microbiology 37: 54-60.
Friday, D. Tuite, J., and Stroshine, R. 1989. Effect of hybrid and physical damage on mold development and carbon dioxide production during storage of high-temperature and shelled corn. Cereal Chemistry 66: 422-426.
Gajendra, B., Baktavachalam, B.D., Fisher, T.L. Ladics, G.S., Layton, R.J., Locke, M., Schmidt, J., Anderson, J.A., Weber, N.N., Herman, R.A., and Evans, S.L. 2015. Transgenic maize event TC1507: Global status of food, feed, and environmental safety, GM Crops & Food, 6(2): 80-102. DOI: 10.1080/21645698.2015.1054093.
Garland T., and Reagor, J. 2001. Chronic canine aflatoxicosis and management of an epidemic. In Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium, ed. W DeKoe, R Samson, H van Egmond, et al. Wageningne, Netherlands: Ponsen and Looven 231–236pp.
Garman, H., and Jewett, H.H. 1914. The life-history and habits of the corn-ear worm (Chloridea obsoleta). Kentucky Agricultural Experimental Station Bulletin 187: 513– 591.
Gassmann, A.J. Petzold-Maxwell, J.L., Clifton, E.H., Dunbar, M.W., Hoffmann, A.M., Ingber, D.A., and Keweshan, R.S. 2014. Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize. Proceedings of the National Academy of Sciences of the United States of America 111: 5141–5146.
Giles, K.L., Hellmich, R.L., Iverson, C.T. and Lewis, L.C. 2000. Effects of Transgenic Bacillus thuringiensis Maize Grain on B. thuringiensis-Susceptible Plodia interpunctella (Lepidoptera: Pyralidae). Stored-product and Quarantine Entomology 93(3): 1011- 1016.
Golob, P.N., Kutukwa, A. Devereau, R.E., Bartosik, and Rodriguez, J.C. 2004. Chapter two: Maize. In Crop Post-Harvest: Science and Technology. Vol. 2. R. Hodges, and G. Farrell, eds. Ames, Iowa. Blackwell Publishing Ltd.
Gould, F. 1998. Sustainability of transgenic insecticidal cultivars: Integrating pest genetics and ecology. Annual Review of Entomology 43: 701-726.
Halpin, C. 2005. Gene stacking in transgenic plants–the challenge for 21st century plant biotechnology. Plant Biotechnology Journal 3: 141-155. doi.org/10.1111/j.1467 7652.2004.00113.
Hambleton, L.D. 1979. Spontaneous heating of grain. In: Grain Storage. Technical Publication Trust: Perth, Australia. 94107pp. 36
Hammond, B., Campbell, K., Pilcher, C., Robinson, A., Melcion, D., Cahagnier, B., et al. 2003. Reduction of fumonisin mycotoxins in Bt corn. The Toxicologist 2003;72(S-1): abstract 1217.
Hanley, A.M., Wilkins, T.M., Sedlacek, J.D. 2004. Cry1Ab and Cry9C transgenic corn hybrid effects on laboratory populations of Indianmeal moth (Lepidoptera: Pyralidae) and Angoumois grain moth (Lepidoptera: Gelechiidae). Journal of Entomological Science 39:514–24.
Hansen, L., Lövei, G., and Szekacs, A. 2013. Survival and development of a stored-product pest, Sitophilus zeamais (Coleoptera: Curculionidae), and its natural enemy, the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae), on transgenic Bt maize. Pest Management Science 69(5) doi:10.1002/ps.3410.
Hardke, J.T., Leonard, B.R., Huang, F., Jackson, R.E. 2011. Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis Cry proteins. Crop Protection 30(2): 168–72; dx.doi.org/10.1016/j.cropro.2010.10.005.
Headrick, J.M. 2006. Application of multiple approaches toward reducing aflatoxin contamination of corn grain. Proceedings of the 2006 Annual Multi-Crop USDA Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshop, 16–18 Oct 2006. Ft Worth Texas. p. 33.
Hedayati, M.T., Pasqualotto, A.C., Warn, P.A., Bowyer, P., Denning, D.W. 2007. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153:1677– 92.
Herrero, S., Oppert, B., and Ferre, J., 2001. Different mechanisms of resistance to Bacillus thuringiensis toxins in the Indianmeal moth. Applied and Environmental Microbiology 67: 1085–1089.
Hockensmith, P.E., Devine, T.L. Legg, D.E. and Rodriguez, J.G. 1986. Energy consumption and food utilization of the Indianmeal moth (Lepidoptera: Pyralidea) on the different corn genotypes. Journal of Kansas Entomological Society 59:598-603.
Hodges, R. 2011. Missing Food: The Case of Postharvest Grain Losses in Sub-Saharan Africa The International Bank for Reconstruction and Development / The World Bank. Report No. 60371-AFR.
Horn, B.W. 2003. Ecology and population biology of aflatoxigenic fungi in soil. Journal of Toxicology 22(2-3): 351-379. 37
Horn, B.W., Greene, R.L., Dorner, J.W. 1995. Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia. Applied Environmental Microbioogy 61: 2472–2475.
Horn, B.W., Moore, G.G., and Carbone, I. 2009. Sexual reproduction in Aspergillus flavus. Mycologia 101: 423-29.
Howe, R.W. 1972. Insects attacking seeds during storage. 247-300pp. In: Seed Biology. T.T. Kozlowski (Ed.). Vol 3. Insect and Seed Collection, Storage, Testing and Certification. Academic Press: New York. 368pp.
ISAAA. 2017. Global Status of Commercialized Biotech/GM Crops in 2017: Biotech Crop Adoption Surges as Economic Benefits Accumulate in 22 Years. ISAAA Brief No. 53. ISAAA: Ithaca, NY.
Ishiwatari, S. 1901. On a kind of severe flacherie (sotto disease). Dainihon Sanshi Kaiho 114: 5.
Jacobs, K. and D. Calvin. 1988. Weevils on stored grain. Entomological Notes. Pennsylvania State University. http://ento.psu.edu/extension/factsheets/weevils-on-stored- grain
James, C. 2006. Global Status of Commercialized Biotech/GM Crops: 2006. ISAAA Brief 35. International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY.
Jian, F., and Jayas, D.S. 2012. The ecosystem approach to grain storage. Agricultural Research 1(2): 148-156.
Johnson, D. E., Oppert, B., and McGaughey, W.H. 1998. Spore coat protein synergizes Bacillus thuringiensis crystal toxicity for the Indianmeal moth (Plodia interpunctella). Current Microbiology 36: 278-282.
Jones, R.K., Duncan, H.E., Payne, G.A., and Leonard, K.J. 1980. Factors influencing infection by Aspergillus flavus in silk-inoculated corn. Plant Disease 64: 859–863.
Jouanin, L., Bonade-Bottino, M., Girard, C., Morrot, G., and Giband, M. 1998. Transgenic plants for insect resistance. Plant Science 131: 1-11.
Kaliyan, N., Carrillo, M.A., Vance. M., William W., and Cannon, C.A. 2005. Indian meal moth survivability in stored corn with different levels of broken kernels. The Great Lakes Entomologist 38:2 Art. 6. available at: https://scholar.valpo.edu/tgle/vol38/iss2/6
Kankolongo, M.A., Hell, K., Nawa, I.N. 2009. Assessment for fungal, mycotoxin and insect spoilage in maize stored for human consumption in Zambia. Journal of the Science and Food Agriculture 89: 1366-1375. Doi:10.1002/jsfa.3596. 38
Keyl, A.C. 1978. Aflatoxicosis in cattle. In: Wyllie TD, Morehouse LG editors. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, Volume 2. Marcel Dekker, New York; p. 9-27.
Klich, M.A. 1986. Mycoflora of cotton seed from the southern United States: a three year study of distribution and frequency. Mycologia. 78: 706-712.
Klich, M.A. 2007. Aspergillus flavus: the major producer of aflatoxin. Molecular Plant Pathology 8: 713–22.
Koziel, M.G., Beland, G. L. , Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L., Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, M. R., Merlin, E., Rhodes, R., Warren, G.W., Wright, M., and Evola, S.V. 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Biotechnology 11: 194-200.
Kramer, K.J., Morgan, T.D., Throne, J.E., Dowell, F.E., Bailey, M., and Howard, J.A. 2000. Transgenic avidin maize is resistant to storage insect pests. Nature Biotechnology 18: 670-674.
Krishnan, S., Manavathu, E.K., and Candrasekar, P.H. 2009. Aspergillus flavus: an emerging non fumigatus Aspergillus species of significance. Mycoses 52: 206-22.
Kurtz, H.J., Mirocha, C.J. 1978. Zearalenone (F2) induced estrogenic syndrome in swine. In: Wyllie TD, Morehouse LG, editors. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, Volume 2. Marcel Dekker, New York. p. 1256–64.
La Prade, J.C. and Manwiller, A. 1977. Relation of insect damage, vector, and hybrid reaction to aflatoxin B1 recovery from field corn. Phytopathology 67:544-547.
Lacey, J., Hill, S.T. and Edwards, M.A. 1980. Microorganisms in stored grains: their enumeration and significance. Tropical Stored Products Information 39: 19-33.
Lee, S.E., and Campbell, B.C. 2000. In vitro metabolism of aflatoxin B1 by larvae of navel orangeworm, Amyelois transitella (Walker) (Insecta, Lepidoptera, Pyralidae) and codlingmoth, Cydia pomonella (L.) (Insecta, Lepidoptera, Tortricidae). Archives of Insect Biochemistry and Physiology 45: 166-174.
Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., George, L, et al. 2005. Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya. Environmental Health Perspectives 113:1763–67
Lillehoj, E.B., Kwolek, W.F.K., Fennel, D.I. and M.S. Milburn. 1975. Aflatoxin incidence and association with bright greenish-yellow fluorescence and insect damage in a limited survey of freshly harvest high-moisture corn. Cereal Chemistry 52:403-412. 39
Loewer, O.J., Bridges, T., and Bucklin, R. 1994. On-farm drying and storage systems. American Society of Agricultural Engineers.
Long K., Ghazali, H.M., Ariff, A., Man, Y.C., and Bucke, C. 1998. Substrate preference of mycelium-bound lipase from a strain of Aspergillus flavus Link. Biotechnology Letters 20(4): 369-372.
Longstaff, B.C. 1981. Biology of the grain pest species of the genus Sitophilus (Coleoptera: Curculionidae): a critical review. Protection Ecology 3(2): 83-130.
Lynch, R.E., Plaisted, W.D. and Warnick, D. 1999. Evaluation of transgenic sweet corn hybrids expressing CrylA(b) toxin for resistance to corn earworm and fall armyworm (Lepidoptera: Noctuidae). Journal of Economic Entomology 92: 246-252.
MacKenzie, S.A., Lamb, I., Schmidt, J., Deege, L., Morrisey, M.J., Harper, M., et al. 2007. Thirteen week feeding study with transgenic maize grain containing event DAS- 01507–1 in Sprague Dawley rats. Food and Chemical Toxicology 45:551–62.
Magg, T., Melchinger, A.E., Klein, D., and Bohn M. 2002. Relationship between European corn borer resistance and concentration of mycotoxins produced by Fusarium spp. in grains of transgenic Bt maize hybrids, their isogenic counterparts, and commercial varieties. Plant Breeding: Zeitschrift fur Pflanzenzuchtu 121(2):146–54.
Mannaa, M., and Kim, K.D. 2017. Influence of temperature and water activity on deleterious fungi and mycotoxin production during grain storage. Mycobiology 45(4): 240-254. doi:10.5941/MYCO.2017.45.4.240.
Markham, R. H., Bosque-Perez, N. A., Borgemeister, C., and Meikle, W. G. 1994. Developing pest management strategies for Sitophilus zeamais and Prostephanus truncates in the tropics. FAO Plant Protection Bulletin 42(3): 97-116. Retrieved from http://agris.fao.org/agris-search/search.do?recordID=XF9657657.
Mason, L. 2003. Insects and mites. In: Food Plant Sanitation. Hui, Y.H., Bruinsma, B.L., Gorham, J.R., Nip, W.K., Tong, P.S., Ventresca, P. (Eds.). Marcel Dekker: New York. 293-315pp.
Matsumura, F. and Knight, S.G. 1967. Toxicity and chemo-sterilising activity of aflatoxin against insects. Journal of Economic Entomology 60: 871-872.
Maupin, L.M., Clements, M.J., Walker, S.L., and White DG. 2001. Effects of Cry1Ab on Aspergillus ear rot in commercial corn hybrids. Phytopathology 91: S59.
Mbata, G.N. 1990. Suitability of maize varieties for the oviposition and development of Plodia interpunctella (Hubner) (Lepidoptera: Pyralidea). Tropical Pest Management. 36: 122-127. 40
McGaughey, W.H. 1985. Insect resistance to the biological insecticide Bacillus thuringiensis. Science 229: 193-195.
McGaughey, W.H. 1994. Problems of insect resistance to Bacillus thuringiensis. Agriculture, Ecosystems & Environment 49: 95-102.
McGaughey, W.H., and Johnson, D.E. 1992. Indianmeal moth (Lepidoptera: Pyralidae) resistant to different strains and mixtures of Bacillus thuringiensis. Journal of Economic Entomology 85: 1594-1600.
McGaughey, W.H., and Johnson, D.E. 1994. Influence of crystal protein composition of Bacillus thuringiensis strains on cross-resistance in Indianmeal moths (Lepidoptera: Pyralidea). Journal of Economic Entomology 87: 535-539.
McMillian, W.W., Wilson, E.M., Widstrom, N.W. and Perkins, Q.D. 1980. Effects of aflatoxin BI and G1 on those insect pests of maize. Journal of Economic Entomology 73: 26-28.
Medina, A., Gilbert, M.K., Mack, B.M., O’Brian, G.R., Rodriguez, A., Bhatnagar, D., Payne, G., Magan, N. 2017. Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production. International Journal of Food Microbiology 256: 36-44.
Mendelsohn, M. 1998. Biopesticide fact sheet: Bacillus thuringiensis subspecies kurstaki Cry1A(c) delta endotoxin and the genetic material necessary for its production in corn. EPA 730-F-98-015.
Mendelsohn, M. 1999. Biopesticide fact sheet: Bacillus thuringiensis Cry1A(b) delta endotoxin and the genetic material necessary for its production in corn. EPA 730-F- 99-004.
Miller, J.D. 1995. Fungi and mycotoxins in grain: implications for stored product research. Journal of Stored Product Research 31:1-6.
Miller, J.D., Simon, J.W., Blackwell, B.A., Greenhalgh, R., and Taylor A. 2001. Deoxynivalenol: a 25 year perspective on a trichothecene of agricultural importance. In: Summerell BA, Leslie JF, Backhouse D, Bryden WL, Burgess LW, editors. Fusarium. APS Press, St. Paul, MN. p. 310–20.
Moellenbeck, D.J., Peters, M.L., Bing, J.W., Rouse, J.R., Higgins, L.S., Sims, L., Nevshemal, T., Marshall, L. 2001. Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms. Nature Biotechnology 19(7): 668-72.
Mohandass, S., Arthur, F.H., Zhu, K.Y., Throne, J.E. 2007. Biology and management of Plodia interpunctella (Lepidoptera: Pyralidae) in stored products. Publications from USDA- ARS/UNL Faculty. http://digitalcommons.unl.edu/usdaarsfacpub/1981. 41
Moreno-Martinez, E. and Christensen, C.M. 1971. Differences among lines and varieties of maize in susceptibility to damage by storage fungi. Phytopathology 61:1498-1500.
Mousa, W., Ghazali, F.M., Jinap, S., Ghazali, H.M., and Radu, S. 2013. Modeling growth rate and assessing aflatoxins production by Aspergillus flavus as a function of water activity and temperature on polished and brown rice. Journal of Food Science 78: 56- 63.
Mullen, M.A., and Arbogast, R.T. 1977. Influence of substrate on oviposition by 2 species of stored product moths. Environmental Entomology 6: 641-644.
Munkvold, G., Hellmich, R., and Rice, L. 1999. Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and non-transgenic hybrids. Plant Disease 83: 130-138.
Munkvold, G.P. 2003. Epidemiology of Fusarium diseases and their mycotoxins in maize ears. European Journal of Plant Pathology 109: 705-713.
Munkvold, G.P. and Hellmich, R.L. 2000. Genetically modified, insect resistant maize: implications for management of ear and stalk diseases. Plant Health Progress, Plant Management Network. Available from: URL: http://www.plantmanagementnet work. org /pub/php/review/maize.
Muzemu, S., Chitamba, J., and Goto, S. 2013. Screening of stored maize (Zea mays L.) varieties grain for tolerance against maize weevil, Sitophilus zeamais (Motsch.). International Journal of Plant Biology & Research 3: 17-22.
Nansen, C., Phillips, T.W., Parajulee, M.N., and Franqui, R.A., 2004. Comparison of direct and indirect sampling procedures for Plodia interpunctella in a maize storage facility. Journal of Stored Products Research 40: 151-168.
Ngindu, A., Johnson, B.K., Kenya, P.R., Ngira, J.A., and Ocheng, D.M. 1982. Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenta. Lancet 319(8285):1346–48.
Niu, G., Siegel, J., Schuler, M.A., Berenbaum, M.R. 2009.Comparative toxicity of mycotoxins to navel orangeworm (Amyelois transitella) and corn earworm (Helicoverpa zea). Journal of Chemical Ecology 35: 951-957.
Njoroge, A., Affognon, H., Mutungi, C., Manono, J., Lamuka, P., and Murdock, L. 2014. Triple bag hermetic storage delivers a lethal punch to Prostephanus truncates (Horn)(Coleoptera: Bostrichidae) in stored maize. Journal of Stored Products Research 58: 12-19. 42
O’Brian, G.R., Georgianna, D.R., Wilkinson, J.R., Abbas, H.K., Wu, J., Bhatnagar, D., Cleveland, T.E., Nierman, W., and Payne, G.A. 2007. Effect of elevated temperature on gene expression and aflatoxin biosynthesis. Mycologia 90: 232–239.
Odvody, G.N., Chilcutt, C.F., Parker, R.D., Benedict, J.H. 2000. Aflatoxin and insect response of near-isogenic Bt and non-Bt commercial corn hybrids in south Texas. In: Proceedings Aflatoxin/Fumonisin Workshop. Robens, JF. (Ed.). Beltsville MD: USDA- ARS.
Palma, L., Muñoz, D., Berry, C., Murillo, J., and Caballero, P. 2014. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6: 3296-3325. doi:10.3390/toxins6123296.
Papst, C., Utz, H.F., Melchinger, A.E., Eder, J., Magg, T., Klein, D., et al. 2005. Mycotoxins produced by Fusarium spp. in isogenic Bt vs non-Bt maize hybrids under European corn borer pressure. Agronomy Journal 97:219-24.
Payne, G.A., Cassel, D.K. and Adkins, C.R. 1985. Reduction of aflatoxin levels in maize due to irrigation and tillage. Phytopathology 75: 1283-1283.
Phillips, T.W., Berbert, R.C., and Cuperus, G.W. 2000. Post-harvest integrated pest management. In: Encyclopedia of Food Science and Technology. Francis, F.J. (Ed.). 2nd ed. Wiley Inc.: New York, 2690-2701pp.
Phillips, T.W., Strand, M.R., 1994. Larval secretions and food odors affect orientation in female Plodia interpunctella. Entomologia Experimentalis et Applicata 71: 185-192.
Pingali, P.L., and Pandey, S. 2001. Meeting world maize needs: technology opportunities and priorities for the public sector. In: World Maize Facts and Trends Meeting World Maize Needs: Technological Opportunities and Priorities for the Public Sector. Pingali, P.L. (Ed.). CIMMYT: Mexico City. CIMMYT 1999-2000.
Pittendrigh, B.R., Huesing, J.E., Shade, R.E. and Murdock L.L. 1997. Effects of lectins, CRY1A/CRY1B Bt δ-endotoxin, PAPA, protease and α-amylase inhibitors on the development of the rice weevil, Sitophilus oryzae, using an artificial seed bioassay. Entomologia Experimentalis et Applicata 82: 201-211.
Plumlee, K.H., and Galey, F.D. 1994. Neurotoxic mycotoxins: a review of fungal toxins that cause neurological disease in large animals. Journal of Veterinary Internal Medicine 8: 49-54.
Probst, C., Schulthess, F., and Cotty, P.J. 2010. Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays). Journal of Applied Microbiology 108:600–10. 43
Rees, D. 2004. Insects of Stored Products. CSIRO Publishing, Collingwood, Victoria: Australia. 181p.
Riley, C.V. 1882. The boll-worm alias corn-worm (Heliothis armigera Hü bn.) order Lepidoptera; family Noctuidae. In: Report of the Commissioner of Agriculture for the years 1881 - 1882. Washington Printing Office. pp. 145–152.
Ross, P.F., Rice, L.G., Osweiler, G.D., Nelson, P.E., Richard, J.L., and Wilson, T.M. 1992. A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates. Mycopathologia 117:109- 14.
Roucan-Kane, M., and Gray, A. 2009. The U.S. seed industry: An exploration of statistics highlighting the economic activity of the U.S. row crop seed industry. Working Paper #09-08. Dept. of Agricultural Economics. Purdue University.
Rubens J., and Cardwell, K.F. 2003. The cost of mycotoxin management in the United States. Journal of Toxicology 22(2-3): 139-152.
Sanchis, V., and Magan, N. 2004. Environmental conditions affecting mycotoxins. In: Mycotoxins in food: Detection and control. Magan, N., Olsen, M., (Eds.). Cambridge: Woodhead Publishing Ltd. 74-189 pp.
Sauer, D.B., Meronuck, R.A., and Christensen, C.M. 1992. Microflora. In: Storage of cereal grains and their products, D.B. Sauer (Ed). American Association of Cereal Chemists 312-340pp.
Schaafsma, A.W., Hooker, D.C., Baute, T.S., and Illincic-Tamburic, L. 2002. Effect of Bt-corn hybrids on deoxynivalenol content in grain at harvest. Plant Disease 86(10):1123-6.
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., and Dean, D.H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews 62: 775-806.
Schnepf, H.E., Lee, S., Dojillo, J., Burmeister, P., Fencil, K., Morera, L., Nygaard, L., Narva, K.E., and Wolt, J.D. 2005. Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections. Applied and Environmental Microbiology 71: 1765-1774.
Schulthess, F., Cardwell, K.F., Gounou, S. 2002. The effect of endophytic Fusarium verticillioides on infestation of two maize varieties by lepidopterous stemborers and coleopteran grain feeders. Phytopathology 92: 120-128. 44
Sedlacek, J.D., Komaravalli, S.R., Hanley, A.M., Price, B.D., and Davis, P.M. 2001. Life History Attributes of Indian Meal Moth (Lepidoptera: Pyralidae) and Angoumois Grain Moth (Lepidoptera: Gelechiidae) Reared on Transgenic Corn Kernels. Stored-Product and Quarantine Entomology 94(2): 586-592.
Setamou, M., Cardwell, K.F., Schulthess, F., and Hell, K. 1998. Effect of insect damage to maize ears, with special reference to Mussidia nigrivenella (lepidoptera: pyralydae), on Aspergillus flavus (deuteromycetes: monoliales) infection and aflatoxin production in maize before harvest in the Republic of Benin. Journal of Economic Entomology 91: 433-438.
Sinha R.N., and Wallace, H.A.H. 1965. Ecology of a fungus-induced hot spot in stored grain. Canadian Journal of Plant Science 45:48-59.
Sinha, R. N. 1984. Effects of weevil (Coleoptera: Curculionidae) infestation on abiotic and biotic quality of stored wheat. Journal of Economic Entomology 77: 1483-1488.
Sinha, R.N. 1971. Fungus as food for some stored product insects. Journal of Economic Entomology 64: 3-6.
Smale, M., Derek, B., and Thom, J. 2011. Maize revolutions in Sub-Saharan Africa. Policy research working paper 5659. The World Bank. Development research group. Agriculture and rural development team. Available at: http://wwwwds.worldbank.org/servlet/WDSContentServer/WDSP/IB/2011/05/1 1/000158349_20110511144001/Rendered/PDF/WPS5659.pdf
Sobek, E.A., and Munkvold, G.P. 1999. European corn borer (Lepidoptera: Pyralidae) larvae as vectors of Fusarium moniliforme, causing kernel rot and symptomless infection of maize kernels. Journal of Economic Entomology 92(3):503-509.
Sone, J. 2001. Mold growth in maize storage as affected by compound factors: Different levels of maize weevils, broken corn and foreign materials, and moisture contents. Journal of Asia-Pacific Entomology 4(1): 17-21.
Sorenson, W.G., Hesseltine, C.W., Shotwell, O.L. 1967. Effect of temperature on production of aflatoxin on rice by Aspergillus flavus. Mycopathologia et mycologia applicate 33: 49-55.
St. Leger, R. J., Screen, S. E., Shams-Pirzadeh, B. 2000. Lack of host specialization in Aspergillus flavus. Applied Environmental Microbiology 66:320-324.
Steinbach, W.J. 2005. Pediatric aspergillosis. Diagnosis and treatment differences in children. The Pediatric Infectious Disease Journal 24:358–64. 45
Stenske, K.A., Smith, J.R., Newman, S.J., Newman, L.B., and Kirk, C.A. 2006. Aflatoxicosis in dogs and dealing with suspected contaminated commercial foods. Journal of the American Veterinary Medical Association 228: 1686-1691.
Strosnider, H., Azziz-Baumgartner, E., Banziger, M., Bhat, R.V., Breiman, R., et al. 2006. Workgroup report: public health strategies for reducing aflatoxin exposure in developing countries. Environmental Health Perspectives 114:1898–903.
Suleiman, R., Rosentrater, K.A., and Bern, C.J. 2015. Evaluation of maize weevils Sitophilus zeamais Motschulsky infestation on seven varieties of maize. Journal of Stored Products Research 64: 97-102.
Summer, W.A., Harein, P.K., and Subramanyam, B.H. 1988. Malathion resistance in larvae of some southern Minnesota populations of the Indianmeal moth, Plodia interpunctella (Lepidoptera: Spyralidea), infesting bulk-stored shelled corn. Great Lakes Entomology 21: 133-137.
Tabashnik, B.E., and W.H. McGaughey. 1994. Resistance risk assessment for single and multiple insecticides: response of Indianmeal moth (Lepidoptera: Pyralidea) to Bacillus thuringiensis. Journal of Economic Entomology 87: 834-841.
Thompson, G.D., Dalmacio, S.C., Criador IV, A.R., Alvarez, E.R., Hechanova, R.F. 2010. Field performance of TC1507 transgenic corn hybrids against Asian corn borer in the Philippines. Philippine Agricultural Scientist 93(4): 375-83.
Tilak, R., Singh, A., Maurya, O.P., Chandra, A., and Tilak, V. 2010. Mycotic keratitis in India: a five-year retrospecive study. Journal of Infection in Developing Countries 4:171–74.
Tzanakakis, M.E., 1959. An ecological study of the Indian meal moth, Plodia interpunctella, with emphasis on diapause. Hilgardia 29: 205-246.
U.S. Environmental Protection Agency. 2000. Publ. No. EPA731-F-95–004 (U.S. Govt. Printing Office, Washington, DC. http://www.epa.gov/scipoly/sap/2000/october/.
U.S. Environmental Protection Agency. 2004. Review of Product Characterization and Expression Analysis for Registration of Bt Cry34/35AB1 Construct PHP17662 Corn. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough Ph.D. to M. Mendelsohn dated December 6, 2004.
U.S. Environmental Protection Agency. 2006. Modified Cry3A protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8(006509) Fact Sheet https://www3.epa.gov/pesticides/chem_search/reg_ actions /registration/fs_PC- 006509_18-May-10.pdf. 46
U.S. Environmental Protection Agency. 2007. Biopesticide Registration Action Document Modified Cry3A Protein and The Genetic Material Necessary for Its Production (Via Elements of Pzm26) In Event MIR604 Corn SYN-IR604-8 (March 2007).
U.S. Environmental Protection Agency. 2009. Biopesticides Registration Action Document Bacillus thuringiensis Vip3Aa20 Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements of Vector pNOV1300) in Event MIR162 Maize (OECD Unique Identifier: SYN-IR162-4z) PC Code: 006599.
U.S. Environmental Protection Agency. 2010a. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1Ab and Cry1F Corn (Updated September 2010).
U.S. Environmental Protection Agency. 2010b. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (plasmid insert PHP 17662) in Event DAS-59122-7 corn (006490) Fact Sheet (http://ceragmc. org/files/cera/GmCropDatabase/docs/decdocs/05-354-002.pdf)]
Van Wyk, J.H., Hodson, A.C. and Christensen, C.M. 1959. Microflora associated with the confused flour beetle, Tribolium confusum. Annals of Entomological Society of America. 52: 452-463.
Wang, H., Eyun, S., Arora, K., Tan, S.Y., Gandra, P., Moriyama, E., Khajuria, C., Jurzenski, J., Li, H., Donahue, M., Narva, K. and Siegfried, B. 2017. patterns of gene expression in western corn rootworm (Diabrotica virgifera virgifera) neonates, challenged with Cry34Ab1, Cry35Ab1 and Cry34/35Ab1, based on next-generation sequencing. Toxins 9 (124): 1-13.
Wiatrak, P.J., Wright, D.L., Marois, J.J., and Wilson, D. 2005. Influence of planting date on aflatoxin accumulation in Bt, non-Bt, and tropical non-Bt hybrids. Agronomy Journal 97:440–5.
Widstrom, N.W., Sparks, A.N., Lillehoj, E.B. and W.F. Kwolek. 1975. Aflatoxin production and lepidopteran insect injury in corn in Georgia. Journal of Economic Entomology 68: 855-856.
Williams, R. J., and Macdonald, D. 1983. Grain molds in the tropics: Problems and importance. Annual Review of Phytopathology 21(1): 153-178.
Wilson, S., and Desmarchelier, J. 1994. Aeration according to seed wet-bulb temperature. Journal of Stored Products Research 30(1): 45-60.
Windham, G.L., Williams, W.P., and Davis, F.M. 1999. Effects of the southern corn borer on Aspergillus flavus kernel infection and aflatoxin accumulation in maize hybrids. Plant Disease 83: 535–540. 47
World Health Organization. 1993. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 56. Lyon: IARC Press. 397-444pp.
World Health Organization. 2018. Aflatoxins. REF. No.: WHO/NMM/FOS/RMA/18.1 available at https://www.who.int/foodsafety/FSDigest_Aflatoxins_EN.pdf
Wu, F.E. 2004. Explaining public resistance to genetically modified corn: An analysis of the distribution of benefits and risks. Risk Analysis: An International Journal 24:715- 726.
Wu, F.E. 2006. Mycotoxin reduction in Bt corn: potential economic, health, and regulatory impacts. Transgenic Res. 15:277–89
Wu, F.E. 2007. Bt corn and impact on mycotoxins. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2, No. 060.
Yeh, M.Y. 1979. Dissemination of toxigenic fungi in shelled corn by Ahasverus advena and Tribolium confusum. University of Minnesota: Minneapolis, MN. 71p.
Yu, C.G., Mullins, M.A., Warren, G.W., Koziel, M.G., and Estruch, J.J. 1997. The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Applied Environmental Microbiology 63(2): 532–536.
Zhao, X, Wang, D., Fields, P.G., Li, H. 2018. Effect of aflatoxin B 1 on development, survival and fecundity of Ahasverus advena (Waltl). Journal of Stored Products Research 77: 225-230.
48
CHAPTER 2. INTERACTIONS BETWEEN ASPERGILLUS FLAVUS AND STORED-GRAIN INSECTS IN CONVENTIONAL AND TRANSGENIC MAIZE
Abstract
Fungal colonization and mycotoxin contamination are chronic problems that can compromise grain quality and safety in stored maize. Aflatoxins, associated with Aspergillus flavus Link infection, are the most economically important mycotoxins. Insect damage is a major factor that predisposes grain to infection by mycotoxigenic fungi. Indianmeal moth
(Plodia interpunctella Hübner) and maize weevil (Sitophilus zeamais Motschulsky) are lepidopteran and coleopteran stored-grain insects, respectively, that can cause considerable damage in stored maize. The effect of Indianmeal moth or maize weevil infestations on A. flavus colonization and vice versa in non-Bt and Bt maize hybrids with lepidopteran and coleopteran events were evaluated in this study. After 28 days of storage at 32°C and 80-85% relative humidity, the presence of Indianmeal moth or maize weevil did not enhance A. flavus colonization in non-Bt or Bt hybrids. No Indianmeal moths or maize weevils survived in grain of Bt hybrids with lepidopteran or coleopteran resistance genes, respectively. Aflatoxin levels in the 106 A. flavus inoculated non-Bt grain increased significantly in the presence of Indianmeal moth or maize weevil (p ≤ 0.01), but aflatoxins in the Bt hybrids were unaffected by insect infestation. A. flavus caused increased mortality, reduced survivorship, and lower growth indices of both insects, hence, limiting their feeding activity in the inoculated grain compared to the uninoculated. As a result, damage
(p ≤ 0.0001) and grain weight loss (p ≤ 0.01) were significantly higher in the uninoculated non-Bt hybrid. In the transgenic hybrids, no damage and grain weight loss were observed regardless of the presence or absence of A. flavus, due to 100% mortality of insects. A. flavus 49 effects on insects were evident only in non-Bt hybrid and insect infestation only enhanced aflatoxin contamination in the absence of Bt protection. Mycotoxin reduction in Bt maize has been well-studied in the field but not in storage. This study demonstrated that Bt protection was effective against stored-grain insects Indianmeal moth and maize weevil.
The use of Bt hybrids can mitigate the risk of A. flavus infection and aflatoxin contamination related to stored-grain insects.
Introduction
Grain quality of maize is primarily reduced by the presence of storage molds and stored-grain insects. Grains stored at high temperature and moisture conditions are more susceptible to mold and insect infestations. Modern grain handling and storage facilities allow controlled storage conditions but in areas where artificial drying facilities are not available or accessible, maintaining grain at safe moisture levels remains a challenge. Maize grain can be stored safely at 18oC or less; temperatures between 21-27oC are the most conducive for the majority of storage molds (Sauer et al., 1992). Aspergillus flavus, a storage mold that causes rotting of maize kernels and aflatoxin contamination, grows optimally at
o 30-35 C and 0.95 water activity (aw) in storage. These storage conditions favorable for A. flavus growth are common in the tropics such as countries in Asia and Sub-Saharan Africa, making aflatoxin contamination a significant chronic problem. A. flavus is a major producer of aflatoxin B1 which is a potent naturally occurring carcinogen of the liver, kidney, lungs, and colon in humans and animals (WHO, 1993). In humans, ingestion of aflatoxin contaminated food products over time may lead to liver cancer, stunted growth in children, and suppressed immune system (Turner et al., 2003; Cardwell et al., 2004). Repeated 50 outbreaks of acute aflatoxicosis have been reported in Kenya, causing death of hundreds of people (Probst et al., 2010). In animals, aflatoxins can cause liver and kidney damage, reduced weight gain, and lower productivity. To prevent risks associated with ingestion of aflatoxins, allowable threshold levels were established. In the US, the Food and Drug
Administration (FDA) determined an action level of 20 ppb in all products, except milk, for human consumption. For animal consumption of maize, action levels were 100 ppb for breeding beef cattle, swine, and mature poultry; 200 ppb for finishing swine; and up to 300 ppb for mature beef cattle (Smith, 1997; Amaike & Keller, 2011).
Insect feeding predisposes grain to A. flavus infection and aflatoxin contamination by creating points of entry for colonization (Diener et al., 1987). It was shown that aflatoxin levels were higher in maize grain infested with maize weevils (Sitophilus zeamais
Motchulsky) and A. flavus than in grains with A. flavus only (Beti et al., 1995). Maize weevil and Indianmeal moth (Plodia interpunctella Hübner) are coleopteran and lepidopteran insect pests, respectively, commonly found attacking maize grain in many countries throughout the world, including the tropics. Indianmeal moth is an external feeder that causes damage in the grain during its larval phase. While the larvae feed, silken threads are formed causing webbing, and their frass and cast skin gives off an unpleasant odor in the infested grain (Mohandass et al., 2007). Maize weevil, on the other hand, is an internal feeder that starts consuming the grain as larva from within the kernel. Unlike Indianmeal moth, both the larval and adult phase of maize weevil actively feed on the grain. Maize weevil is capable of consuming kernels completely in storage and producing considerable amount of grain dust at the same time. The feeding damage incurred by Indianmeal moth or maize weevil reduces the overall nutritive and market value of the grain. 51
The presence of Bt proteins in maize, as a result of deterring insect activity, has been shown to indirectly reduce mycotoxin contamination in the field. Several field studies have shown significantly reduced fumonisin levels in Bt hybrids compared to non-Bt isolines
(Munkvold et al., 1999; Dowd, 2001; Bowers et al., 2013). However, field results have shown variable results for aflatoxins. Significant aflatoxin reduction was observed in Bt maize compared to non-Bt in some years (Williams et al., 2002; Headrick, 2006; Abbas et al., 2006); while there are also years where aflatoxin levels of the Bt and non-Bt maize were not different (Odvody et al., 2000; Buntin et al., 2001; Wiatrak et al., 2005). Studies on the Bt maize-mycotoxin relationship have been entirely in the field and any effect of Bt proteins on mycotoxin levels have not been demonstrated in post-harvest conditions. Bt proteins such as Cry1Ab, mCry3A, Cry1Fa2 in Bt11, Mir604 and TC1507 events, respectively, are expressed in the grain and have around 1-2 ng/mg mean expression levels in the grain by dry weight (EPA, 2010b, 2007). The presence of Bt proteins in the grain suggests the possibility of insect protection during grain storage. The few studies conducted under post-harvest conditions showed that Bt maize was effective against stored-grain insects such as Indianmeal moth and angoumois grain moth (Sitotroga cerealella) (Giles et al., 2000; Sedlacek et al., 2001; Hanley et al., 2004).
Insect control is undeniably an important component in managing storage molds and a key strategy in mitigating mycotoxin risks in maize. At present, Bt maize provides the most effective way to suppress insect pest populations and has been proven to indirectly reduce mycotoxin levels in the field; this warrants a study on their potential impact on stored-grain insects and aflatoxin contamination in storage. In this study, the effects of
Indianmeal moth or maize weevil infestation on A. flavus and vice versa were reported; as 52 well as the impact of these insect-fungus interactions on grain quality in terms of damage, grain weight loss, and aflatoxin contamination. Insect-A. flavus interactions were compared in non-Bt and Bt maize hybrids with lepidopteran or coleopteran events.
Materials and Methods
In this experiment, four maize hybrids were used to study the interactions of A. flavus and Indianmeal moth. One is a conventional hybrid that served as the control and the other hybrids express different Bt events; all have herbicide-tolerance genes, others express single Bt genes or in combination (Table 1). All the hybrids received were untreated, i.e., the seeds were clean and not subjected to any physical, biological or chemical treatments prior to packaging. A lateral flow strip test was performed on the non-
Bt hybrids to detect presence of Bt proteins such as Cry1Ab, Cry1F, Cry3A, and Vip3Aa. The limit of detection for this protein-based assay is 1.0%.
A. flavus isolate. There were three A. flavus isolates initially tested, all were isolated from maize in Iowa. After single-spore isolation, the isolates were cultured in ground maize to test for aflatoxin production. Ground maize was autoclaved for 20mins for two consecutive days and weighed 25 grams into 50ml conical tubes. These tubes with ground maize were then inoculated with 2.5 ml spore suspension (106) of A. flavus isolate. About 2 weeks after inoculation, when ground maize was completely colonized, cultures were analyzed for aflatoxin levels at the National Center for Agricultural Utilization Research, USDA-ARS,
Peoria, IL. Out of the three isolates tested, only one produced aflatoxin. This aflatoxin- producing isolate was selected to be used for the subsequent experiments.
53
Grain sample preparation. Grain samples were kept in cold storage to maintain a moisture content of 12-13% until use. Before inoculation of A. flavus, samples were conditioned for 2-3 days at 32°C and 80-85% RH to reach a moisture content of 16-17%.
After inoculation, 200 grams from each hybrid were placed into 473-ml jars, occupying approximately 50% by volume of the jars with enough headspace present for insect movement and air flow and then covered with wire-mesh (80US/tyler) centered canning rings and filter paper. This would help maintain the desired moisture content of the grain in favor of A. flavus growth at the same time allowing ample air necessary for insect survival. Each of the storage jars with maize was considered as an experimental unit.
Inoculation of A. flavus. A. flavus cultures were prepared from stock culture on anhydrous silica gel transferred onto potato dextrose agar (PDA) plates and incubated at room temperature for at least 10 days. A. flavus spore suspensions were prepared by dislodging the spores from plates using sterile distilled water and a glass L-rod, filtering the suspension with sterile mesh cloth, and counting spores using hemocytometer to make necessary adjustments of the spore concentration. Suspension were standardized to 106 spores/ml for the first trial and 105 spores/ml for the second trial. Maize kernels were coated with spore suspension using a Wintersteiger Hege 11 liquid seed treater (Figure 1).
Batches of 2,500 grams grain were treated with 18ml of the spore suspension while grain was rotating in the seed treater for 2mins. Based on a preliminary trial, grain moisture content was not significantly changed after treatment of 18ml water, and this amount also was enough to evenly cover 2,500 grams of grain as determined using a water with colorant.
54
Indianmeal moth infestation. Indianmeal moth eggs and the standard rearing diet formulation were obtained from the Stored Products Entomology Laboratory, Dept. of
Entomology and Plant Pathology, Oklahoma State University, OK. The diet is mainly composed of yellow corn meal (1 lb) and 15% all mash eggs crumbs for laying hens (2 quarts), chick starter and grower crumbles (2 quarts) and glycerin (1.5 quarts). The eggs were transferred to 473-ml jars with the rearing diet and incubated at 32°C and 80-85%
RH until larvae emerged and reached 2-3 days old. The larvae were separated using USA standard No. 20 and 40 mesh sieves to separate larvae from the diet. This was done in a well-lighted and warm space to encourage movement of larvae without too much agitating.
Fifty (50) larvae were carefully picked using a paint brush and transferred directly into each jar with the treated grain. Corrugated cardboard (2.5 by 30 cm) were placed on top of grain introduced with Indianmeal moth larvae to provide suitable pupation sites.
Maize weevil infestation. A total of 20 unsexed maize weevil was introduced into each jar containing the test hybrids. Adults were collected from rearing jars with maize grain maintained at 27°C and 60-65% relative humidity from the Department of Agricultural and
Biosystems Engineering, Iowa State University. Individual adults were separated from the rearing substrate by sieving using a USA standard No. 20 mesh and those that were collected in the bottom pan were aspirated into clean tubes to separate 20 individual adults. After collection, adults were transferred immediately into jars containing the treated grain.
Storage conditions. Grain samples were kept in the Percival environmental chamber
(Percival Scientific Inc., Boone, IA) set at 32±2oC (89.6±2oF), 80-85% RH to maintain 16- 55
17% moisture content. The chamber was previously calibrated and RH readings verified using concentrated salt solution with known RH at a given temperature. The jars remained undisturbed throughout the storage period in the dark, for a period of 28 days, enough to allow one insect generation only. This number of days for storage is based on the reported minimum life cycle for Indianmeal moth and maize weevil under optimum conditions.
Experimental design and data collection. The experiment was established in a completely randomized design with the following treatments for each of the 4 hybrids: 1) control treatment - grain treated with 18 ml water only; 2) grain treated with A. flavus spore suspension; 3) grain treated with water and infested with Indianmeal moth; 4) grain treated with water and infested with maize weevils; 5) grain treated with A. flavus spore suspension and infested with Indianmeal moth; and 6) grain treated with A. flavus spore suspension and infested with maize weevils. Four replicate jars were prepared for each treatment, giving a total of 96 jars. Each jar was regarded as an experimental unit. Jars were randomized within shelves for each replicate. Temperature and RH profiles were monitored using WatchDog Data Loggers A-series (Percival Scientific Inc.) throughout the storage period. Moisture content of grain samples was measured using the Grain Analyzer
Computer (GAC) 2000 Dickey-john Corporation, Auburn, IL). In the end of storage, all stages of Indianmeal moth or maize weevil were collected and counted: adults were carefully picked and transferred to glass plates; larvae were separated through sieving; and
Indianmeal moth pupae formed in jars or in corrugated board were also collected. Kernels were then examined thoroughly for feeding damage of Indianmeal moth. Damaged and undamaged kernels were both counted and weighed to compute for the percent damage and percent weight loss (Adams & Schulten, 1978) expressed as: 56
Number of damaged kernels per jar % ������ = � 100 Total number of kernels per jar
(Wu x Nd) − (Nu x Wd) % ����ℎ� ���� = � 100 Wu x (�� + ��)
Where, Wu = weight of undamaged grains; Wd = weight of damaged grains; Nu = number of undamaged grains; Nd = number of undamaged grains.
For insect measurements, percentage mortality, percentage survivorship, and growth index (Zhang et al., 1993) were computed using the following formulae:
no. of dead individuals % ��������� = � 100 initial no. of population
% ���������ℎ�� = ( . . . ) x 100 .
∑( No. of individuals in a stage x stage no. ) + (no. of dead individual � (stage number − 1) �����ℎ ����� = total no. of individuals counted x highest attainable stage
Moisture content of the grain and fungal population were determined before and after storage. A. flavus population was measured by spread plate method on Aspergillus selective media and a general media for molds. Details are described in the next section.
A. flavus population count. To eliminate surface contaminants, kernels were submerged into 0.5% sodium hypochlorite (NaOH) solution for 3 mins and rinsed thrice in sterile distilled water. The kernels were allowed to dry overnight before milling. Milling was done in the fume hood using the Romer Analytical Sampling Mill. The entire 200-gram sample per jar was milled and after every treatment the mill was cleaned thoroughly to avoid cross contamination. One gram from each sample was used as stock for serial dilution. From the diluted suspension, 0.1ml aliquot was aseptically transferred to 57 previously prepared Aspergillus flavus parasiticus agar (AFPA) and Dichloran Rose Bengal
Chloramphenicol (DRBC) plates. The suspension was then spread evenly on the surface of the media using an L-shaped rod and the plates were incubated at room temperature for 3-
5 days. Three replicates were prepared for each dilution. Single colonies were counted and the dilution that showed 10-100 colonies were used to calculate the colony forming units per ml (cfu/ml) by multiplying the average number of colonies with the dilution factor divided by the volume plated.
Aflatoxin analysis. Samples were sent and analyzed in USDA-ARS, Peoria, Illinois for aflatoxin levels determined by Liquid Chromatography-Mass Spectrometry (LC-
MS) system. Details of the procedures are provided below. From the remaining milled sample, 10 grams was obtained to be extracted with 10 mL of a mixture (1:1) of acetonitrile and water. The extract was filtered and analyzed by LC-MS (Dionex Ultimate UPLC connected to a ThermoFisher QExactive MS). Ten (10) uL of the extract was injected on a
C18 column (2 mm x 150 mm) and eluted with a 0.6 mL/min flow of methanol and water.
The analytes were eluted by applying a 40-95% aqueous methanol gradient over 6 min.
Detection of the aflatoxins was done by looking for the protonated ions [M+H]+ of the toxins and quantitation by comparison of the toxin MS response to toxin standards. For highly infested samples, more extraction solvent was used than normal and samples were diluted to 1:100 for better MS results.
Statistical analysis. All statistical analyses were performed with R-Studio statistical software version 1.1.383 – package (emmeans). The responses are grain weight loss, percent damage, percent mortality, percent survivorship, growth index, aflatoxin levels, and colony counts (CFU/gram). Means of each response were compared by analysis of 58 variance (ANOVA) and multiple comparison test using Tukey’s least significant difference
(LSD) at p = 0.05.
Results
Indianmeal moth infestation
NK1284-GT (GT), a non-Bt hybrid, was used first to compare A. flavus and storage insect interactions prior to using the organic non-Bt hybrid (R19) as the non-Bt control hybrid for this study. Indianmeal moth and maize weevil adult survivorship on non-Bt GT were only 1-3% and 3-10%, respectively. This is a very low survival rate considering that the test grain is a non-Bt hybrid. Indianmeal moth introduced as eggs failed to develop into larvae or adult on non-Bt GT and if introduced as larvae, adult survivorship became 2-5%.
Survivorship to larvae was 8-10% if non-Bt grain was larvae infested and with 1 g milled grain. However, no adult survivorship and feeding damage on the whole grain was observed. Percentage of damaged kernels was not significantly different in maize stored entirely in the dark or with 12-hr light (Figure 1).
Storage at 32oC and 70-75% relative humidity
Storage conditions of 32oC and 70-75% relative humidity were maintained to reach
14-15% grain moisture contents of non-Bt GT and all the Bt hybrids. Only 23.5% of
Indianmeal moth and 5% maize weevil survived by the end of the storage period (Figure
2). Survivorship of Indianmeal moth and maize weevil were significantly lower in A. flavus inoculated non-Bt grain (p ≤ 0.001). The damage by Indianmeal moth (2.07%) on non-Bt grain was higher (p ≤ 0.001) in non-inoculated than with A. flavus (1.37%). The same trend was not seen in weevil infested grain with only less 1% damage in both treatments. No A. 59 flavus colonies were recovered across all treatments including non-Bt. Conidiation of
Aspergillus was observed at the surface of kernel tips during storage but no A. flavus colonies were isolated after the kernels were surface sterilized and plated in AFPA for CFU count. Instead, Fusarium spp. at a level of 7.6x102 CFU/g were recovered from non- inoculated non-Bt infested with Indianmeal moth only. Even on DRBC, a selective medium for molds, only Fusarium spp. were recovered in that same treatment (6.0x103 CFU/g). In all the Bt hybrids, no Indianmeal moth and maize weevil remained alive after 4 weeks of storage and damage was around 0.1-0.7% only and not significantly different with or without A. flavus (p > 0.05) (Figure 2).
Storage at 32oC and 80-85% relative humidity
Grain moisture content was 16-17% at 32oC and 80-85% relative humidity storage conditions. Larval survivorship of Indianmeal moth was 24% on non-inoculated non-Bt and higher on the inoculated non-Bt grain with 34.5% survivorship (p ≤ 0.01). Feeding damage of Indianmeal moth reached an average of 9% and 10% on non-inoculated and inoculated non-Bt, respectively (p > 0.19). For the maize weevil, survivorship on grain without A. flavus was 1.6% and higher on grain inoculated with A. flavus with 5.8% survivorship but not statistically different (p > 0.43). The Bt hybrid with only lepidopteran events (Cry1Ab x
Cry1F x Vip3Aa) had 17% survivorship of maize weevil on A. flavus uninoculated grain and only 3% with A. flavus (p ≤ 0.01). Damage was also significantly different in these treatments with 1.7% damage on non-inoculated grain and 0.4% damage on inoculated grain (p ≤ 0.01). No living larvae, pupa, or adults of Indianmeal moth were seen on the Bt hybrids with lepidopteran events. All maize weevil adults were also dead after the storage period on Bt hybrids with coleopteran events. All grains, non-Bt or Bt hybrids, have some 60 levels of A. flavus with the highest count in the non-Bt GT. Indianmeal moth infested non-Bt
GT without A. flavus had an average of 1.8x104 CFU/g and 3.2x104 CFU/g on the A. flavus inoculated treatment. With maize weevil infestation and no A. flavus, colony count was
2.4x104 CFU/g and without maize weevil only A. flavus contamination was 1x104 CFU/g.
The control treatment had an average of 2.1x104 CFU/g and the grain without insects but A. flavus only had 1.5x104 CFU/g. However, median A. flavus levels across treatments within each hybrid were not significantly different from each other both in the non-Bt and Bt hybrids based on the natural log-transformed data. Initial moisture contents of the grain were between 16-17% but it decreased to 14.5-15.5% by the end of the storage period
(Figure 3).
Comparison of Indianmeal moth colonies OK and KS
Survivorship and damage of Indianmeal moth colonies from Oklahoma State
University (OK) and USDA Kansas (KS) were compared on several non-Bt hybrids other than NK1284-GT (GT); i.e., 34C17 (C17) and 09R19 (R19) which are organic hybrids, and
NK0760-GT (60GT). Indianmeal moth was reared on grains maintained at 16-17% moisture content. Among these four hybrids, OK or KS Indianmeal moths had the highest survivorship on R19 ranging from 38-104% and damage of 6-25% (p ≤ 0.0001), while the rest were not significantly different from each other whether OK or KS colonies were infested as eggs or larvae. Survivorship on C17 ranged from 19-83%; on GT it was 1-27%, and; on 60GT was 9-45% on whole kernels. Damage ranged from 2-8% for C17, 0.1-4% for
GT and 1-8% for 60GT.
Comparing the two colonies on R19, survivorship and damage were higher with OK than KS both in egg and larvae infestation on whole kernels (p ≤ 0.0001). OK infested as 61 larvae had higher survivorship (104%) than eggs (57%) (p ≤ 0.01), but the damage on kernels was not significantly different between the eggs (24%) and larvae (19%).
Survivorship was also 104% when 10% cracked maize was incorporated with the whole kernels (p > 0.99). The damage was lowest in 10% cracked maize (14% damage) than in larvae or egg infested on whole kernels with 19% and 24% damage, respectively, although they are not statistically different (Figure 5). Visual inspection of the damage by OK and KS larvae showed larger and more evident feeding sites on R19 (Figure 6). The average size of
OK colonies was10mm for the larvae and 8 mm for pupa and adults. KS colonies larvae have an average of 6mm and 5mm for both pupa and adult (Figure 7). Life cycle duration from larvae to adult of OK and KS colonies were 16-17 days and 10-12 days, respectively.
Presence of Bt proteins in the non-Bt hybrids
Because of the poor survivorship of insects on non-Bt hybrids, presence of Bt contamination was suspected. The different non-Bt hybrids used in the preliminary experiments were tested for possible contamination of Bt proteins. Lateral flow strip testing for several lepidopteran events was conducted at the Seed Trait Lab, Seed Science
Center. Percentages were given based on the estimated contamination values using the strip manufacturer’s scanner. GT had 1.10% Cry1Ab; 60-GT had 1.30% Cry1Ab, 3.50% cp4epsps, 0.38% Cry34 and 1% Cry2A; C17 had 1.50% Cry1Ab, 2.70% cp4epsps, 0.38%
Cry38b and 2.20% Cry2A. Only R19 had less than the limit of detection for all the proteins tested.
62
Background levels of R19 and NK1284 Bt hybrids
Storage molds found on the grains were Cladosporium, Aspergillus, Penicillium, and
Fusarium. Penicillium was found in all grains except R19 in both DRBC and AFPA selective media. Fusarium was the second most predominant mold isolated in R19 and all NK1284 Bt hybrids -3000GT, -3122, and -3220 then Cladosporium which was only found on hybrids
3122 and 3220. Although AFPA was a selective medium for A. flavus and A. parasiticus, characteristic orange colonies were detected only in R19; for the remaining hybrids, other molds proliferated instead (Figure 9). Only Fusarium on DRBC and Aspergillus on AFPA were found in R19. No aflatoxins B1, B2, G1 or G2 were found in all the hybrids except in
R19 which had 50 ppb of total aflatoxin. Fumonisins were detected in non-Bt GT with a total of 5.13 ppb, 3000GT with 1.56 ppb, 3122 with 2.52 ppb, and 3220 with 1.92 ppb
(Table 3).
Storage conditions for A. flavus-insect interactions
In the preliminary result of storing grains at 32oC and 80-85% relative humidity, grains reached a moisture content of 16-17%. A. flavus colonies were isolated after storage but CFU/g were not significantly different among treatments and moisture content dropped to 14.5-15.5%. To help maintain 16-17% grain moisture content, jar lids were placed with filter paper below the screen cover. Adding filter paper in the cover did not affect the development of insects. Since the development and damage of Indianmeal moth were similar in the presence or absence of light, dark conditions were used in this study to mimic storage conditions in reality where grains are stored in containers that are likely covered and not exposed to light. The organic non-Bt R19 was shown to be totally free of Bt proteins and survivorship of Indianmeal moth was the highest, therefore, R19 was used as 63 the non-Bt control for the subsequent experiments. Also, OK colonies were used for further experiments since the comparison between OK and KS colonies of Indianmeal moth showed OK colonies being superior to KS colonies in terms of development and damage.
Whole kernels of maize were used since adding milled grain or 10% cracked maize with the whole kernels to supposedly give Indianmeal moth a head start for development resulted in having no or less damage on the whole kernels because larvae only fed on the milled or cracked grain. The same colonies of maize weevil were used since survivorship was relatively normal in R19.
Survivorship per growth stage in the non-Bt grain
Survivorship was determined per developmental stage of Indianmeal moth and maize weevil. Live Indianmeal moth larvae were found in the non-Bt grain R19 without A. flavus after 4 weeks of storage at 32oC and 80-85% relative humidity. Larval survivorship was higher on grains without A. flavus; no live larvae were found on A. flavus inoculated grains regardless of the inoculum level of 106 or 105 spores/ml (p ≤ 0.01 and ≤ 0.0001, respectively). In R19 inoculated with 106 spores/ml, 4% remained as intact pupae and only
2% of the initial population developed into adults. In the absence of A. flavus, adult survivorship reached 9%. When the inoculum level was decreased to 105 spores/ml, 60% larvae from the initial population were alive, only 1.5% were intact pupae and 26% reached the adult stage. There was no significant difference between with or without A. flavus on pupal and adult survivorship. Comparing the inoculum levels showed significantly increased adult survivorship at 105 spores/ml inoculum level (p ≤ 0.0001).
Survivorship of maize weevil larva and adult were higher in the absence of A. flavus
(p ≤ 0.0001) in both inoculum level except in the larval stage observed in the 105 inoculated 64 grain. Significantly more adults survived (114%) in 105 inoculum compared to the mean survivorship of maize weevil adults in 106 inoculum which was only 15% (p ≤ 0.0001)
(Figure 10).
Mortality and growth indices in non-Bt and Bt hybrids
From the same experiment as above, mortality was compared in each hybrid and growth index was used as a measure of insect development from the initial population of larvae to adults for Indianmeal moth and from adults to a new generation of larvae for maize weevil (Figure 11). In Bt hybrids with lepidopteran events expressing Cry1Ab,
Cry1Ab x Cry1F, and Cry1Ab x Cry1F x Vip3Aa proteins, the mortality of Indianmeal was all
100% at the end of storage period. Growth indices of Indianmeal moth were all zero in the presence of the lepidopteran events with or without A. flavus. Mortality in the non-Bt control was 73% without A. flavus and reached 94% in grains inoculated with 106 spores/ml (p ≤ 0.0001). Growth index was 24% without A. flavus and only 7% in the presence 106 A. flavus (p ≤ 0.0001). Similarly, growth index reached 70% and 42% in the non-inoculated and 105 inoculated grains, respectively.
The presence of coleopteran events expressing mCry3A and mCry3A x
Cry34A/35Ab1 proteins resulted in 100% mortality of maize weevil and no growth index value. In the same hybrid without any coleopteran event but with Cry1Ab x Cry1F x
Vip3Aa, mortality was 93% and 78% in grains treated with 106 A. flavus or without A. flavus, respectively (p ≤ 0.0001). At 105 A. flavus initial inoculum, mortality in Cry1Ab x
Cry1F x Vip3Aa was reduced to 70%. Only 2-7% of the initial population of maize weevil died in the non-Bt control in both trials without A. flavus and 69% on the non-Bt grains with 106 A. flavus initial inoculum. This was reduced to 23% when grains were inoculated 65 with 105 A. flavus. Growth indices were largely higher without the presence of A. flavus in non-Bt and Cry1Ab x Cry1F x Vip3Aa hybrids (p ≤ 0.0001) in the 106 A. flavus trial. Growth index of maize weevil in non-Bt further increased in 105 A. flavus inoculation but not in
Cry1Ab x Cry1F x Vip3Aa.
Damage and grain weight loss
Indianmeal moth damage in A. flavus inoculated grain was 2% in 106 inoculum and
6% without A. flavus (p ≤ 0.0001). In 105 inoculum, percent damage was 8% and without A. flavus damage was 18%. The 106 A. flavus inoculated grain had a lower weight loss of 0.3% compared to the non-inoculated control with 0.7% (p ≤ 0.01). Weight loss in 105 A. flavus inoculated non-Bt was comparable with the non-inoculated non-Bt grain (1.2% and 1.1%, respectively). Damage and weight loss were zero or very low in Cry1Ab, Cry1Ab x Cry1F and Cry1Ab x Cry1F x Vip3Aa.
Maize weevil feeding in the non-inoculated non-Bt grain resulted in 24% damaged kernels with 2.7% weight loss, higher than in 106 inoculated non-Bt with only 7% damaged kernels and 0.86% weight loss (p ≤ 0.0001). Feeding damage was also seen in Cry1Ab x
Cry1F x Vip3Aa but the percentage and weight loss were very low, with a range of 0.7-3% damage and 0.2-0.3% weight loss only with or without A. flavus. Percent damaged kernels increased to 13% in the presence of A. flavus in lower initial inoculum (105) from 7% damaged kernels found in 106 A. flavus treated non-Bt. Only 1-3% damaged kernels were seen on Cry1Ab x Cry1F x Vip3Aa and almost zero weight loss. Kernels with mCry3A and mCry3A x Cry34A/35Ab1 proteins had no reduction in weight and no visible damage on kernels (Figure 12).
66
A. flavus population levels
A. flavus counts in grains treated with 106 initial inoculum had a range of 2.0 x 105 to
3.7 x 106 CFU/gram and the 105 inoculum had 6.7 x102 to 3.4 x103 CFU/gram after the 4 weeks storage period. These levels were significantly higher than the grains treated with water only in the 106 trial (p ≤ 0.0001). Median CFU/g of the natural log-transformed data were used to compare significant differences among the treatments. The presence of insects in non-Bt grain treated with 106 or 105 inoculum did not increase the levels of A. flavus in both non-Bt and Bt hybrids either infested with Indianmeal moth or maize weevil.
Although colony counts were higher in A. flavus only compared to A. flavus + Indianmeal moth treatment, differences were not statistically significant. There was no difference between A. flavus levels across all treatments except in Cry1Ab x Cry1F where A. flavus treated grains had higher colony counts than the control (p ≤ 0.01).
The presence of maize weevil also did not increase A. flavus levels of non-Bt hybrids or hybrids with Cry1Ab x Cry1F x Vip3Aa, mCry3A, and mCry3A x Cry34A/35Ab1 events.
No significant differences of A. flavus levels were observed in the 105 inoculum trial except in mCry3A where colony counts of A. flavus on grains with both A. flavus and maize weevil were higher than with maize weevil only (p ≤ 0.01), and the A. flavus only inoculated mCry3A x Cry34A/35Ab1 had higher A. flavus count than the non-inoculated control (p ≤
0.05). Some Fusarium and bacterial colonies were seen in the uninoculated non-Bt grain but not in the A. flavus inoculated grains (Figure 13).
Aflatoxin contamination levels
Total aflatoxin levels were measured, i.e., AFB1, AFB2, AFG1 and AFG2, but no AFG2 was detected in the trial with 106 inoculum. AFG2 and AFG1 were also not detected in the 67
105 inoculum trial. AFB1 was the most common aflatoxin in these experiments with an average of 220 ppb detected in the non-Bt inoculated control with maize weevil infestation.
In the Indianmeal moth experiment with 106 A. flavus inoculum, aflatoxins in non-Bt inoculated grain infested with Indianmeal moth had an average of 266 ppb, which was significantly higher than without the presence of Indianmeal moth with 170 ppb only (p ≤
0.01). Cry1Ab had only 1.3 and 2.6 ppb in grains with and without Indianmeal moth, respectively. Aflatoxin contamination in Cry1Ab x Cry1F hybrid with only A. flavus (106) was 72 ppb while grain with both A. flavus and Indianmeal moth had only 38 ppb. Cry1Ab x
Cry1F x Vip3A inoculated grain had 144 ppb of aflatoxin without Indianmeal moth and when infested had only 82 ppb. In both hybrids with lepidopteran events, there were no significant differences in aflatoxins between the A. flavus only treatment and the
Indianmeal moth + A. flavus treatment. Aflatoxin contamination was lower in A. flavus only treated non-Bt and Bt hybrids when inoculum level was reduced to 105 (34 ppb) compared to 106 (97 ppb). In A. flavus inoculated Cry1Ab x Cry1F hybrid there was no significant difference between aflatoxin levels with or without Indianmeal moth (Figure 15).
Infestation of maize weevil significantly increased aflatoxins in the A. flavus inoculated non-Bt hybrid, from 170 ppb in the non-infested to 245 ppb in the maize weevil infested treatment (p ≤ 0.01). This was only observed in non-Bt inoculated with 106 A. flavus and not in the 105. Cry1Ab x Cry1F x Vip3Aa had 144 ppb in 106 which did not
5 significantly differ from the levels with maize weevil present (94 ppb). With 10 initial inoculum, Cry1Ab x Cry1F x Vip3Aa (which does not have Coleopteran insect protection) with both A. flavus and maize weevil (86 ppb) had higher aflatoxin contamination compared to the A. flavus only (2.3 ppb) (p ≤ 0.01). Comparing the two hybrids with 68 coleopteran events, mCry3A x Cry34A/35Ab1 had average aflatoxin of 72 ppb and 87 ppb in 106 and 105 A. flavus inoculated grain without maize weevil, respectively, which are both higher than the hybrid with mCry3A only with 3-4 ppb in the 106 and 105 A. flavus only inoculated grain (Figure 15).
Discussion
Interactions between Aspergillus flavus Link and storage insects, Indianmeal moth
(Plodia interpunctella Hübner) and maize weevil (Sitophilus zeamais Motschulsky), varied substantially depending on the initial inoculum level, presence or absence of Bt proteins in the grain, source of insects, and storage conditions. In this study, Indianmeal moth or maize weevil infestations did not enhance A. flavus levels in non-Bt or Bt hybrids. A. flavus inoculation of grain was detrimental to both Indianmeal moth and maize weevil as shown by their reduced survivorship and growth indices in the inoculated non-Bt grain. These effects of A. flavus on insects were only observed in non-Bt grain since no Indianmeal moth or maize weevil survived in Bt hybrids with lepidopteran or coleopteran protection, respectively. Indianmeal moth and maize weevil infestation significantly increased aflatoxin contamination in non-Bt grain inoculated with 106 spores/ml A. flavus, but aflatoxin levels were unaffected by insect infestations in Bt hybrids with the appropriate resistance (coleopteran or lepidopteran).
Insect damage to grain is expected to enhance mold proliferation during the insects’ oviposition, and larval or metabolic activities; however, in this study, insect infestation did not enhance A. flavus levels in grain (Figure 13). In the Bt hybrids, insect infestation did not increase A. flavus levels because the insects died quickly and caused little or no damage. 69
The effect of insect infestations on A. flavus levels should be more informative in the non-Bt grain. However, the presence of A. flavus caused mortality of the insects and deterred feeding (Figure 12), limiting the insects’ opportunity to promote colonization. The high inoculation levels and conducive temperature and RH also allowed for extensive A. flavus colonization in the absence of insects, so the role of insects may have been overshadowed under the conditions of our experiments.
In the A. flavus inoculated non-Bt hybrid, mortality of Indianmeal moth and maize weevil was higher compared to the uninoculated non-Bt control. Reducing the inoculum level of A. flavus from 106 to 105 spores/ml reduced the percent mortality of Indianmeal moth and maize weevil in the inoculated non-Bt. The development of the insects was measured by growth index and results showed lower growth indices of insects infested in the non-Bt hybrid with A. flavus. The detrimental effect of A. flavus to Indianmeal moth and maize weevil was not observed in hybrids with lepidopteran and coleopteran events since the presence of Bt protection resulted in 100% mortality of the insects (Figure 11). Maize weevil larvae numbers in the uninoculated non-Bt were significantly higher than in the inoculated which means that larval development was negatively affected by A. flavus.
Comparable percent survivorship in the uninoculated control of both trials (106 and 105 inoculum) validates that the presence of A. flavus was the main factor that negatively affected the development of maize weevil in the A. flavus treated grain. Inspection under microscope of the dead maize weevil and Indianmeal moth adults, and Indianmeal moth immature pupa showed dense sporulation of A. flavus (Figure 7).
In the colony counts of non-Bt grain without A. flavus inoculation, numerous bacterial colonies were recovered, but these were not observed in the inoculated grain 70
(Figure 14). Possibly the high levels of A. flavus in the grain also inhibited the growth of other microflora during storage.
Bt hybrids with lepidopteran events that express Cry1Ab, Cry1F, and Vip3Aa solely or in combination were hypothesized to deter the development of Indianmeal moth, a lepidopteran stored-grain insect. In previous studies on hybrids with Bt11 as the lepidopteran resistance trait (Giles et al., 2000) Cry1Ab caused decreased survivorship, developmental time, and adult body length of Indianmeal moth. Results of this study confirmed that Bt11 event indeed suppressed the development of Indianmeal moth larvae feeding on grains. Indianmeal moth mortality was 100% in the presence of Cry1Ab (Bt11 event), alone or in combination with other Bt events, and no larvae reached pupal or adult stages (Figure 11).
Maize hybrids expressing proteins mCry3a and Cry34A/35Ab1 were developed to provide resistance to corn rootworm species (Diabrotica spp.), which are coleopteran insects. No coleopteran stored-grain insect has been studied for susceptibility to these proteins. mCry3A of Mir604 and Cry34A/35Ab1 of DAS59122-7 events are expressed in grain (Table 2) and thus potentially effective against maize weevil, which feeds only on kernels. It was shown in this study that mCry3a alone caused 100% mortality of maize weevil adults and prevented any evident signs of feeding or oviposition. Likewise, the hybrid with mCry3a stacked with Cry34A/35Ab1 had the same outcome. Although the
Cry1Ab x Cry1F x Vip3Aa hybrid does not have coleopteran protection, mortality of maize weevil was high in this hybrid and the explanation is not clear (Figure 11).
Insect survivorship correlates to the fraction of damaged kernels. The more individuals surviving or remaining as larvae, higher damage would be expected because 71 each larva continuously feeds unless it has undergone diapause. Damaged kernels and grain weight loss with maize weevil infestation were reduced by A. flavus at both inoculum concentrations, but the reductions were smaller at 105 compared to 106 conidia/ml. Maize weevil fed more voraciously on grain without A. flavus, resulting in an average of 24% damaged kernels with 2.7% weight loss. In untreated maize in the tropics or in susceptible varieties, damage caused by maize weevil may reach up to 20-90% weight loss (Muzemu et al., 2013). Duration of storage in this study was for 28 days only, and higher grain weight loss would be expected if storage period with insect infestation had been longer. In the presence of lepidopteran and coleopteran events, very little or no insect damage and weight loss were observed regardless of the presence or the absence of A. flavus.
Indianmeal moth or maize weevil infestation enhanced aflatoxin contamination only in the absence of Bt protection, i.e., in the non-Bt hybrid or Bt hybrids that lack the appropriate event for lepidopteran or coleopteran resistance. Insect feeding increases the opportunity of infection by mycotoxigenic fungi, hence, indirectly resulting in higher mycotoxin levels. This effect of Indianmeal moth infestation on aflatoxin contamination was observed only in the non-Bt hybrid inoculated with 106 A. flavus and not in the Bt hybrids since the presence of Cry1Ab protein totally inhibited Indianmeal moth activity.
Likewise, increased aflatoxin levels in the presence of maize weevil were only significant in the 106 inoculated non-Bt grain. Maize weevil infestation also enhanced aflatoxin contamination in the Bt hybrid with lepidopteran resistance but no coleopteran resistance.
Similarly, it was shown decades ago that maize kernels infested with A. flavus contaminated maize weevils had higher levels of aflatoxin than A. flavus inoculated maize without the weevils (Beti et al., 1995). The aflatoxin levels detected in the inoculated non-Bt used this 72 study reached at most 245 ppb and 266 ppb with maize weevil and Indianmeal moth infestations, respectively. These are extremely high levels of aflatoxin compared to the allowable threshold of aflatoxin in grain for human and animal consumption. Decreasing the initial inoculum reduced aflatoxin levels with weevil infestation to 18 ppb and with
Indianmeal moth to 19 ppb.
During the initial experiments of this study, Indianmeal moth and maize weevil survivorship were poor in the non-Bt grain. To ensure that the insects have good fitness in maize and that the method of infestation and storage conditions is appropriate to observe insect-fungus interactions, preliminary experiments were conducted. Two different sources of Indianmeal moth larvae, KS and OK, were compared in order to optimize
Indianmeal moth survivorship on the non-Bt grain used in this study. In our preliminary experiments, IMM sourced from OK grew to larger sizes than the KS-sourced IMM at all developmental stages (Figure 7), and subsequently caused more feeding damage (Figure
6). Fitness of larvae is important in evaluating damage on grain and interactions with other factors since only the larval stage of Indianmeal moth feeds on grains. Adding cracked or damaged kernels improved survivorship of Indianmeal moth in maize; however, larval feeding was diverted solely to the cracked or milled portions of the stored grain, and no feeding occurred on the whole kernels for 28 days. Since we sought to estimate damage on whole kernels and weight loss computation also requires counting the number of damaged whole kernels, only whole kernels were used in our main experiments. Also, we observed that introducing 2-3 day-old larvae instead of eggs allowed higher survivorship because they were more capable of chewing intact kernels compared to newly emerged larvae
(Figures 1 and 5). 73
Because of poor survival of both Indianmeal moth and maize weevil on non-Bt hybrids, we tested several non-Bt hybrids for the possible presence of Bt proteins. Lateral flow strip tests revealed that only 1 out of the 6 non-Bt hybrids tested was totally free of kernels with Bt proteins. Traces of Cry1Ab among other Cry proteins were found in all the non-Bt hybrids except in R19. Although none of the protein markers detect coleopteran events, this largely explains the low survivorship of insects on hybrid GT which was the first non-Bt control used in this study. Although the susceptibility of grain to mold colonization and insect infestation varies across maize hybrids because of differences in physiochemical properties (Moreno-Martinez and Christensen, 1971; Friday et al., 1989), the variation observed in the non-Bt hybrids in this study was chiefly because of the Bt proteins present in some of the non-Bt grain. Low level presence (LLP) of genetically modified (GM) traits have been previously reported in conventional maize, at levels generally below the 0.9% GM content threshold set by the European Union to define unintentional GM trait presence (Demeke et al., 2006; Zel et al., 2012; Santa Maria et al.,
2014). The Food and Agriculture Organization (FAO) defined LLP as the unintentional presence of low levels of genetically modified (GM) crops that have been approved in at least one country; whereas, adventitious presence (AP) is the unintentional presence of GM crops that have not been approved in any countries. The unintended low level presence of
GM traits can occur as a result of natural movement of seeds or pollen, or human-mediated activities related with field testing, plant breeding, or seed production.
In determining the storage conditions for this study, it was observed that in 32oC and 70-75% RH storage conditions, only Fusarium spp. and no A. flavus colonies were recovered from surface-disinfested kernels after storage, even though superficial A. flavus 74 growth was observed on the kernel tips. The lack of A. flavus recovery was likely because the RH during storage did not encourage internal colonization of the kernels, which under the given conditions, had an equilibrium moisture content (EMC) of 14-15%. Although this is within the reported range under which A. flavus can colonize maize kernels, it is below the optimal moisture content range for this fungus. A. flavus population counts reported in this study represent the level of A. flavus infection of the grain and not the surface contaminants. Optimal RH conditions for Fusarium spp. to develop in storage are even higher, so it is likely that the Fusarium spp. recovered in this preliminary experiment represent colonization of the kernels that was present before the initiation of the experiment. When grain moisture content was conditioned to reach 16-17% by maintaining storage conditions of 32oC and 80-85% RH, A. flavus colonies were present not only superficially but were also recovered in AFPA selective medium. This storage condition (80-85% RH) favored the growth of A. flavus while restricting the growth of other fungi that are not xerophilic. The characteristic of A. flavus being among the few molds that can predominate in a niche with high temperature and moderately high relative humidity made it feasible to study interactions of A. flavus and stored-grain insects without the growth of other storage fungi, and these conditions represent typical storage conditions in tropical developing countries.
A. flavus-insect interactions during maize storage were assessed under conditions conducive for A. flavus, resulting in relatively high fungal colonization and aflatoxin accumulation in the grain. Effects of A. flavus on Indianmeal moth and maize weevil were consistent in both of the inoculum levels tested. Future experiments may focus on interpreting these interactions at lower levels of A. flavus inoculation. Although the non-Bt 75 hybrid used in the main experiments was not genetically related to the Bt hybrids, the comparisons made in this study regarding the interactions of A. flavus inoculation with insect infestation within hybrids are not affected by genetic differences among hybrids; however, comparisons made among hybrids must be cautiously interpreted because we were not able to obtain uncontaminated non-Bt isolines of the Bt hybrids used in this study.
Lepidopteran and coleopteran events present in the Bt maize hybrids used were undoubtedly effective against the two stored-grain insects, Indianmeal moth and maize weevil, respectively. This is the first study showing the effects of Bt events Mir604 and
DAS59122-7 on maize weevil and demonstrating the impact of Bt events in aflatoxin reduction related to stored-grain insects.
References
Adams, J.M., Schulten, G.G.M., 1978. Loss caused by insects, mites and microorganisms. In: Harris, K.L., Lindbland, C.L. (Eds.), Post-harvest Grain Loss Assessment Methods. American Association of Cereal Chemists, USA, pp. 83-95.
Amaike, S., and Keller, N.P. 2011. Aspergillus flavus. Annual Review of Phytopathology 49(1): 107-133. doi: 10.1146/annurev-phyto-072910-09522.
Abbas, H.K., Zablotowicz, R.M., Bruns, H.A., and Abel, C.A. 2006. Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates. Biocontrol Science and Technology 16:437-449.
Beti, J.A., Phillips, T.W., Smalley, E.B., 1995. Effects of maize weevils (Coleoptera: Curculionidae) on production of aflatoxin B1 by Aspergillus flavus in stored corn. Journal of Economic Entomology 88(6): 1776-1782.
Bowers, E. Hellmich, R., and Munkvold, G. 2013. Vip3Aa and Cry1Ab proteins in maize reduce Fusarium ear rot and fumonisins by deterring kernel injury from multiple Lepidopteran pests. World Mycotoxin Journal 6(2): 127-135.
Buntin, G.D., Lee, R.D., Wilson, D.M., and McPherson, R.M. 2001. Evaluation of YieldGard transgenic resistance for control of fall armyworm and corn earworm (Lepidoptera: Noctuidae) on corn. Florida Entomologist 84:37–42. 76
Cardwell, K.F., and Henry, S.H. 2004. Risk of exposure to mitigation of effects of aflatoxin on human health: a west African example. Journal of Toxicology 23:217-47.
Demeke, T., Perry, D.J. and Scowcroft, W.R. 2006. Adventitious presence of GMOs: Scientific overview for Canadian grains. Canadian Journal of Plant Science 86(1): 1-23.
Diener, U.L., Cole, R.J., Sanders, T.H., Payne, G.A., Lee, L.S., and Klich, M.A. 1987. Epidemiology of aflatoxin formation by Aspergillus flavus. Annual Reviews in Phytopathology. 25: 249–270.
Dowd, P.F. 2001. Biotic and abiotic factors limiting efficacy of Bt corn in indirectly reducing mycotoxin levels in commercial fields. Journal of Economic Entomology 94(5):1067- 74.
Fleurat-Lessard F. 2017. Integrated management of the risks of stored grain spoilage by seedborne fungi and contamination by storage mould mycotoxins: an update. Journal of Stored Products Research 71: 22–40.
Friday, D. Tuite, J., and Stroshine, R. 1989. Effect of hybrid and physical damage on mold development and carbon dioxide production during storage of high-temperature and shelled corn. Cereal Chemistry 66: 422-426.
Giles, K.L., Hellmich, R.L., Iverson, C.T. and Lewis, L.C. 2000. Effects of Transgenic Bacillus thuringiensis Maize Grain on B. thuringiensis-Susceptible Plodia interpunctella (Lepidoptera: Pyralidae). Stored-product and Quarantine Entomology 93(3): 1011- 1016.
Hambleton, L.D. 1979. Spontaneous heating of grain. In: Grain Storage. Technical Publication Trust: Perth, Australia. 94107pp.
Hanley, A.M., Wilkins, T.M., Sedlacek, J.D. 2004. Cry1Ab and Cry9C transgenic corn hybrid effects on laboratory populations of Indianmeal moth (Lepidoptera: Pyralidae) and Angoumois grain moth (Lepidoptera: Gelechiidae). Journal of Entomological Science 39:514–24.
Headrick, J.M. 2006. Application of multiple approaches toward reducing aflatoxin contamination of corn grain. Proceedings of the 2006 Annual Multi-Crop USDA Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshop, 16–18 Oct 2006. Ft Worth Texas. p. 33.
Hockensmith, P.E., Devine, T.L. Legg, D.E. and Rodriguez, J.G. 1986. Energy consumption and food utilization of the Indianmeal moth (Lepidoptera: Pyralidea) on the different corn genotypes. Journal of Kansas Entomological Society 59:598-603. 77
Kaliyan, N., Carrillo, M.A., Vance. M., William W., and Cannon, C.A. 2005. Indian meal moth survivability in stored corn with different levels of broken kernels. The Great Lakes Entomologist 38:2 Art. 6. available at: https://scholar.valpo.edu/tgle/vol38/iss2/6
Klich, M.A. 2007. Aspergillus flavus: the major producer of aflatoxin. Molecular Plant Pathology 8: 713–22.
Lacey, J., Hill, S.T. and Edwards, M.A. 1980. Microorganisms in stored grains: their enumeration and significance. Tropical Stored Products Information 39: 19-33.
Mohandass, S., Arthur, F.H., Zhu, K.Y., Throne, J.E. 2007. Biology and management of Plodia interpunctella (Lepidoptera: Pyralidae) in stored products. Publications from USDA- ARS/UNL Faculty. http://digitalcommons.unl.edu/usdaarsfacpub/1981.
Moreno-Martinez, E. and Christensen, C.M. 1971. Differences among lines and varieties of maize in susceptibility to damage by storage fungi. Phytopathology 61:1498-1500.
Munkvold, G., Hellmich, R., and Rice, L. 1999. Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and non-transgenic hybrids. Plant Disease 83: 130-138.
Muzemu, S., Chitamba, J., and Goto, S. 2013. Screening of stored maize (Zea mays L.) varieties grain for tolerance against maize weevil, Sitophilus zeamais (Motsch.). International Journal of Plant Biology & Research 3: 17-22.
Odvody, G.N., Chilcutt, C.F., Parker, R.D., Benedict, J.H. 2000. Aflatoxin and insect response of near-isogenic Bt and non-Bt commercial corn hybrids in south Texas. In: Proceedings Aflatoxin/Fumonisin Workshop. Robens, JF. (Ed.). Beltsville MD: USDA- ARS.
Probst, C., Schulthess, F., and Cotty, P.J. 2010. Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays). Journal of Applied Microbiology 108:600–10.
Sanchis, V., and Magan, N. 2004. Environmental conditions affecting mycotoxins. In: Mycotoxins in food: Detection and control. Magan, N., Olsen, M., (Eds.). Cambridge: Woodhead Publishing Ltd. 74-189 pp.
Santa-Maria, M.C., Lajo-Morgan, G. and Guardia, L. 2014. Adventitious presence of transgenic events in the maize supply chain in Peru: A case study. Food Control 41: 96-101.
Sauer, D.B., Meronuck, R.A., and Christensen, C.M. 1992. Microflora. In: Storage of cereal grains and their products, D.B. Sauer (Ed). American Association of Cereal Chemists 312-340pp. 78
Sedlacek, J.D., Komaravalli, S.R., Hanley, A.M., Price, B.D., and Davis, P.M. 2001. Life History Attributes of Indian Meal Moth (Lepidoptera: Pyralidae) and Angoumois Grain Moth (Lepidoptera: Gelechiidae) Reared on Transgenic Corn Kernels. Stored-Product and Quarantine Entomology 94(2): 586-592.
Sinha, R.N. 1971. Fungus as food for some stored product insects. Journal of Economic Entomology 64: 3-6.
Smith, J. 1997. Aflatoxins. In: D’Mello, J.P.F. (Ed.), Handbook of Plant and Fungal Toxicants. CRC Press, Boca Raton, pp. 269–285.
Suleiman, R., Rosentrater, K.A., and Bern, C.J. 2015. Evaluation of maize weevils Sitophilus zeamais Motschulsky infestation on seven varieties of maize. Journal of Stored Products Research 64: 97-102.
Turner, P.C. et al. 2003. Modification of immune function through exposure to dietary aflatoxin in Gambian children. Environmental Health Perspectives 111: 217-20.
U.S. Environmental Protection Agency. 2007. Biopesticide Registration Action Document Modified Cry3A Protein and The Genetic Material Necessary for Its Production (Via Elements of Pzm26) In Event MIR604 Corn SYN-IR604-8 (March 2007).
U.S. Environmental Protection Agency. 2010b. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (plasmid insert PHP 17662) in Event DAS-59122-7 corn (006490) Fact Sheet (http://ceragmc. org/files/cera/GmCropDatabase/docs/decdocs/05-354-002.pdf)]
Wiatrak, P.J., Wright, D.L., Marois, J.J., and Wilson, D. 2005. Influence of planting date on aflatoxin accumulation in Bt, non-Bt, and tropical non-Bt hybrids. Agronomy Journal 97:440–5.
Williams, W.P. et al. 2002. Aflatoxin accumulation in conventional and transgenic corn hybrids infested with Southwestern Corn Borer (Lepidoptera: Crambidae). Journal of Agricultural and Urban Entomology 19(4): 227-236.
World Health Organization. 2018. Aflatoxins. REF. No.: WHO/NMM/FOS/RMA/18.1 available at https://www.who.int/foodsafety/FSDigest_Aflatoxins_EN.pdf
Zel, J., Milavec, M., Morisset, D., Plan, D., Eede, G., and Gruden, K. 2012. How to reliably test for GMOs. In: Springer briefs in food, health, and nutrition. How to reliably test for GMOs (pp. 1-95). Springer US.
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Zhang, M., Chaudhuri, S.K., and Kubo, I. 1993. Quantification of insect growth and its use in screening of naturally occurring insect control agents. Journal of Chemical Ecology 19(6). 1109-1118.
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Table 1. Conventional and transgenic maize hybrids evaluated for effects of interactions between A. flavus and Indianmeal moth or maize weevil. Lepidopteran Coleopteran resistance resistance Hybrid Brand Developer GM trait Proteins GM trait Proteins expressed expressed R19 - - - - Blue River Organic Seed NK1284-3000GT Bt11 Cry1Ab Mir604 mCry3A Syngenta Seeds, Inc. NK1284-3122 Bt11 Cry1Ab Mir604 mCry3A Syngenta TC1507 Cry1Fa2 DAS59122-7 Cry34/35Ab1 Seeds, Inc. NK1284-3220 Bt11 Cry1Ab - - Syngenta Mir162 vip3Aa20 Seeds, Inc. TC1507 Cry1Fa2
Table 2. List of Bt proteins evaluated and details of each protein expressed.
Event name Bt Protein Promoter Mean expression Reference expressed levels in grain1 Bt11 Cry1Ab CaMV 35S 2 ng/mg US EPA 2010b Mir604 mCry3A MTLP 1.09 or 1.95 ng/mg US EPA 2007 Mir162 vip3Aa20 ZmUbiInt 14.7 ng/mg US EPA 2009 TC1507 Cry1Fa2 Zea mays 2.2 ng/mg US EPA, 2010b polyubiquitin promoter DAS59122-7 Cry34Ab1 Zea mays ubiquitin 50 ng/mg US EPA 2004b gene promoter Cry35Ab1 Triticum aestivum 1.00 ng/mg peroxidase gene root- US EPA 2004b preferred promoter 1 measured in dry weight
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0 hr light 12 hr light 15 1.0
✱✱ 0.8
% damage 10 ✱ 0.6
0.4 5
% survivorship 0.2
0 0.0
Adult - 0pp Larvae - 0pp Adult -12pp Larvae - 12pp Egg infested Larvae + milled grain Larvae infested damage on kernels Figure 1. IndianmealIndianmeal moth (IMM) survival and damage on non-Bt maize (GT) stored at IMM rearing conditions (25oC and 60-65% RH) with 0-hr (0pp) and 12- hr (12pp) light interval. No larvae or adult were present on the egg infested grain. Larvae infested grain have higher percentage of larvae that developed into adults. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
IMM MW 30 ✱✱✱ 2.5 ✱✱✱ 2.0 % damage 20 1.5 1.0 10 ✱✱✱
Insect only % survivorship 0.5 Insect only Insect + A. flavus Insect + A. flavus 0 0.0
Cry1Ab mCry3A
Non-Bt (GT) Non-Bt (GT) Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3AaCry1Ab x Cry1F x Vip3Aa Figure 2. Maize grain stored for 4 weeks at 32o and 70-75% RH. Grains were infested with Indianmeal moth (IMM) or maize weevil (MW) and were artificially inoculated with A. flavus. Damage is relative to the total number of sampled grains and survivorship accounts for larvae that reached pupal stage and adults. Non-Bt grain used was GT. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001. 82
IMM MW 50 14 ✱✱ 40 12 % damage 10 30 ✱✱ 8 20 6 4 % survivorship ✱✱ Insect only 10 Insect only 2 Insect + A. flavus Insect + A. flavus 0 0
Cry1Ab mCry3A Non-Bt (GT) Non-Bt (GT) Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3AaCry1Ab x Cry1F x Vip3Aa Figure 3. Maize grain stored for 4 weeks at 32o and 80-85% RH. Grains were infested with Indianmeal moth (IMM) or maize weevil (MW) and were artificially inoculated with A. flavus. Damage is relative to the total number of sampled grains and survivorship accounts for larvae that reached pupal stage and adults. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
IMM MW 14 Control 12 A. flavus only 10 Insect only 8 A. flavus + Insect 6 4 2 0 CFU/gram of milled grain (ln)
Cry1Ab mCry3A Non-Bt-IMM Non-Bt-MW Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3AaCry1Ab x Cry1F x Vip3Aa Figure 4. Log colony forming units (CFU) of A. flavus isolated from maize samples stored for 4 weeks at 32o and 80-85% RH. No significant differences of treatments within hybrids (p > 0.05). 83
✱✱✱ ✱ 40 150 ✱✱✱ ✱✱✱✱ ✱✱ 30 100 ✱✱✱ ✱✱✱✱ 20 50 % damage
% survivorship 10
0 C17 R19 GT 60GT 0 C17 R19 GT 60GT OK-eggs-whole OK-larv-whole OK-larv-10% OK-eggs-whole OK-larv-whole OK-larv-10% KS-eggs-whole KS-larv-whole KS-larv-10% KS-eggs-whole KS-larv-whole KS-larv-10% Figure 5. Comparison of the two Indianmeal moth colonies (OK and KS) based on survivorship and percent damaged kernels with different infestation methods on non-Bt hybrids. C17 and R19 are organic non-Bt hybrids; GT (NK1284-GT) and 60GT (NK0760-GT) are transgenic non-Bt hybrids. Eggs or larvae (larv) were introduced into jars containing whole kernels or with whole kernels + 10% cracked maize. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001. Significance codes on top of OK bars compare differences with KS unless otherwise bracketed.
R19 – IMM eggs (OK) R19 – IMM larvae (OK) R19 – IMM larvae (KS)
Figure 6. Feeding damage of IndianmealIndianmeal moth OK and KS colonies on the organic non-Bt R19. Grain infested with eggs have smaller feeding holes compared to when larvae were introduced. Larvae of OK colonies produced more evident damage compared KS colonies.
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A
Figure 7. IndianmealIndianmeal moth (Plodia interpunctella): A) feeding damage on maize by larvae; B) adult and pupa colonized by A. flavus; and C) comparison of two different colonies of IndianmealIndianmeal moth (larger colony was used for this study).
A B
C
Figure 8. Maize weevil (Sitophlius zeamais): A) bored hole - a characteristic feeding damage of adult weevil; B) inside of damaged kernel with evident A. flavus growth; C) stages of maize weevil found in the non-Bt grain.
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DRBC AFPA
100 100 Cladosporium sp.
Aspergillus sp. 80 80 Penicillium sp. 60 60 Fusarium sp.
40 40 20 20
CFU/gram of milled grain 0 0
R19 3122 3220 R19 3122 3220 3000GT 3000GT Figure 9. Background levels of storage molds from the four maize grain hybrids prior to storage under experimental conditions of 32oC and 80-85% RH. Non-Bt grain – R19; Bt grain with lepidopteran/coleopteran events – 3000GT, 3122, 3220. Two media were used for mold counts: dichloran rose bengal chloramphenicol agar (DRBC) – selective medium for most molds and; A. flavus parasiticus agar (AFPA) selective medium for A. flavus and A. parasiticus.
Table 3. Background mycotoxin levels of the four maize grain hybrids prior storage under experimental conditions. Non-Bt grain – R19; grain with lepidopteran/coleopteran events – 3000GT, 3122, 3220. Total aflatoxin (AFB1+AFB2+AFG1+AFG2) and fumonisin (FB1+B2+B3+B4) levels are shown below.
Hybrid Aflatoxin (ppb) Fumonisin (ppb) Non-Bt (R19) 60.69 - Non-Bt (GT) 0.0 5.13 3000GT 0.0 1.56 3122 0.0 2.52 3220 0.0 1.92
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80 6 5 10 inoculum 10 inoculum ✱✱✱✱ without A. flavus
60 with A. flavus
40
✱✱ 20
% IMM survivorship 0
Larva Pupa Adult Larva Pupa Adult
250 6 105 inoculum 10 inoculum ✱✱✱✱ without A. flavus 200 ✱✱✱✱ with A. flavus
150
100 ✱✱✱✱ 50 % MW survivorship 0 Larva Adult Larva Adult
Figure 10. Survivorship of IndianmealIndianmeal moth (IMM) and maize weevil (MW) relative to the number of initial populations on the non-Bt grain (R19). A. flavus was inoculated to the grain at levels of 106 and 105 spores/ml. Only intact pupae were counted. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001. Mean comparison is within each developmental stage.
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IMM only IMM only MW only MW only IMM + A. flavus IMM + A. flavus MW + A. flavus MW + A. flavus 150 80 150 200 106 inoculum 106 inoculum ✱✱✱✱ 60 150 ✱✱✱✱ 100 ✱✱✱✱ 100
✱✱✱✱ 100 ✱✱✱✱ 40 50 50 20 50 growth index ✱✱✱✱ 0 0 0 0 150 200 150 80 ✱✱✱✱ ✱✱✱✱ 105 inoculum 105 inoculum % mortality 60 150 100 100 ✱✱✱✱ 40 100
50 50 20 ✱✱✱ 50
0 0 0 0
Cry1Ab mCry3A Non-Bt (R19) Non-Bt (R19) Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa
Figure 11. Mortality and growth index of IndianmealIndianmeal moth (IMM) and maize weevil (MW) grown on maize grain stored at 32oC and 80-85% RH. Grains were inoculated with 106 and 105 spores/ml of A. flavus. Mortality is relative to the total number individuals counted after storage and growth index accounts for the surviving individuals per insect developmental stage. Treatment mean comparison is within hybrids. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
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IMM only IMM only MW only MW only IMM + A. flavus IMM + A. flavus MW + A. flavus MW + A. flavus 8 1.0 30 5.0 ✱✱✱✱ 106 inoculum 106 inoculum ✱✱✱✱ 25 ✱✱ 0.8 4.0 6 20 ✱✱✱ 0.6 3.0 4 15 0.4 2.0 10 2 0.2 5 1.0 % weight loss
0 0.0 0 0.0 25 1.5 25 1.0 105 inoculum 105 inoculum % damage ✱✱✱✱ 20 1.2 20 ✱✱✱✱ 0.8
15 0.9 15 0.6
10 0.6 10 0.4
✱✱✱✱ 5 0.3 5 0.2
0 0.0 0 0.0
Cry1Ab mCry3A
Non-Bt (R19) Non-Bt (R19) Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa
Figure 12. Percentage of kernels damaged by insects and weight loss (fresh weight basis) of maize grain stored at 32oC and 80-85% RH. Grains were inoculated with 106 and 105 spores/ml of A. flavus and subsequently infested with Indianmeal moth (IMM) and maize weevil (MW). Treatment mean comparison is within hybrids. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
89
Control IMM only Control MW only A. flavus only IMM + A. flavus A. flavus only MW + A. flavus 20 20 106 inoculum 105 inoculum 106 inoculum 105 inoculum
✱✱✱ ✱✱✱✱ 15 ✱✱✱ ✱✱✱✱ ✱✱✱✱ 15 ✱✱ ✱ ✱✱✱✱ ✱✱✱ ✱✱✱
10 ✱✱ 10 ✱✱ ✱ ✱ 5 5
CFU/gram of milled grain (ln) CFU/gram of milled grain (ln) 0 0
Cry1Ab Cry1Ab mCry3A mCry3A Non-Bt (R19) Non-Bt (R19) Non-Bt (R19) Non-Bt (R19) Cry1Ab x Cry1F Cry1Ab x Cry1F
mCry3A x Cry34/35Ab1 mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa
Figure 13. A. flavus levels measured as colony forming units (CFU) on AFPA selective media on maize grains infested with Indianmeal moth (left) and maize weevil (right). Treatment mean comparison is within hybrids; significance codes on top of bars with A. flavus compare differences with no A. flavus treatment unless otherwise bracketed. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
E
F Figure 14. A. flavus colonies (orange) on AFPA from milled non-Bt (R19) grain: A) control; B) A. flavus inoculated; C) IMM only; D) A. flavus + IMM infested; E) MW only; and, F) A. flavus + MW. Fusarium sp. and bacterial colonies was also be seen on plates A-E. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
90
400 ✱✱ 106 inoculum 105 inoculum Control
A. flavus only 300 IMM only 200 IMM + A. flavus
100
parts per billion (ppb) 0
Cry1Ab Cry1Ab
Non-Bt (R19) Non-Bt (R19) Cry1Ab x Cry1F Cry1Ab x Cry1F
Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa
300 ✱✱ 106 inoculum 105 inoculum Control
250 A. flavus only
200 MW only ✱✱ 150 MW + A. flavus
100
50 parts per billion (ppb) 0
mCry3A mCry3A
Non-Bt (R19) Non-Bt (R19)
mCry3A x Cry34/35Ab1 mCry3A x Cry34/35Ab1 Cry1Ab x Cry1F x Vip3Aa Cry1Ab x Cry1F x Vip3Aa
Figure 15. Total aflatoxin (B1+B2+G1) levels of maize grain stored for 4 weeks under 32oC and 80-85% RH. Aflatoxin level prior to storage was zero except in the non-Bt with 61ppb background. No Aflatoxin G2 was detected in 106 inoculum; and in 105 inoculum both G1 and G2 were not detected. Treatment mean comparison is within hybrids. Significance codes: ‘*’ p ≤ 0.05, ‘**’ p ≤ 0.01, ‘***’ p ≤ 0.001, and ‘****’ p ≤ 0.0001.
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CHAPTER 3. GENERAL CONCLUSIONS
The main objective of this study is the determine the interactions between an aflatoxin producing strain of Aspergillus flavus and the lepidopteran and coleopteran stored-grain insects Indianmeal moth and maize weevil, respectively, in Bt and non-Bt maize under controlled storage conditions. In A. flavus inoculated grain, particularly at 106 inoculum level, has a detrimental effect to insect development causing increased mortality, reduced survivorship, and growth indices of Indianmeal moth and maize weevil. The suppressed insect activity in the inoculated non-Bt resulted in less insect feeding, hence, fewer damaged kernels and lower grain weight loss were observed in the non-Bt grain inoculated with A. flavus.
The presence of Indianmeal moth or maize weevil did not significantly promote A. flavus colonization in both Bt and non-Bt grain because the insects’ opportunity to promote colonization was hindered, as a result of A. flavus and Bt proteins causing suppressed activity of the insects. In all transgenic maize expressing Bt proteins Cry1Ab, Vip3Aa, and
Cry1F solely or in combination, 100% mortality were observed regardless of the presence or absence of A. flavus. The same was observed with maize weevil infested in grains with mCry3a and Cry34A/35Ab proteins. A complete cycle and reproduction of Indianmeal moth and maize weevil was only observed in the non-Bt grain.
Infestation of Indianmeal moth and maize weevil increased aflatoxin levels in the non-Bt hybrid inoculated with 106 A. flavus. This increase in aflatoxin contamination was also observed in the presence of maize weevil in the Bt hybrid without coleopteran protection. The use of Bt hybrids can mitigate mycotoxin risks in stored maize by providing 92 the most effective way to suppress insect pest populations. This has been known and proven in the field, but this is the first study to demonstrate the impact of Bt protection to aflatoxin contamination related to stored-grain insects Indianmeal moth and maize weevil.
The research conducted also provides the first evidence of Mir604 and DAS59122-7 events expressing mCry3A and Cry34A/35Ab1, respectively, effectiveness against maize weevil.
The results of this study contributed to the understanding of A. flavus effect on the two commonly found stored-grain insect in the tropics where A. flavus infection and the associated aflatoxin contamination has always been a problem. More importantly, this study demonstrated the relation of insect suppression provided by the Bt hybrids to aflatoxin contamination in an environment that greatly favors A. flavus infection in storage.
These results are particularly valuable in tropical developing countries where keeping maize at safe moisture levels to prevent aflatoxin contamination during storage remains a challenge.