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1995 Diallel Analyses of Percent Kernel Infection by Aspergillus Flavus and Ear Moisture Loss Rate in Maize. Yudong Zhang Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Zhang, Yudong, "Diallel Analyses of Percent Kernel Infection by Aspergillus Flavus and Ear Moisture Loss Rate in Maize." (1995). LSU Historical Dissertations and Theses. 6148. https://digitalcommons.lsu.edu/gradschool_disstheses/6148

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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 DIALLEL ANALYSES OF PERCENT KERNEL INFECTION BY ASPERGILLUS FLA VUS AND EAR MOISTURE LOSS RATE IN MAIZE

A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Agronomy

by Yudong Zhang B.S.. Nanjing Agricultural University, 1982 M.S..Nanjing Agricultural University. 1985 December 1995 UMI Number: 96X8338

UMI Microform 9618338 Copyright 1996, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 ACKNOWLEDGMENTS

The author is indebted to his major professor. Dr. Manjit S. Kang for his

great support, advice, and help in his more than 3-year study at Louisiana State

University. He would like to give his most sincere gratitude to Dr. Manjit S.

Kang for his input, advice, and guidance throughout the course of this study. The

author also would like to express his appreciation to his minor Professor, Dr.

Kenneth W. Paxton, and other committee members. Dr. Freddie A. Martin, Dr.

Don R. Labonte, Dr. Gerald O. Myers, and Dr. David York for their helpful

suggestions, input, and editorial advice.

The author acknowledges the contributions of his co-worker, Mr. Robert

Magari, for his help in field experiment and data collection. The author also gives thanks to the Agronomy Department for its support and help.

I give my most sincere appreciation to my wife, Haiyan Chen, for her

love, help, understanding, patience, anti encouragement.

ii TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... ii

LIST OF TABLES ...... v

ABSTRACT...... vi

INTRODUCTION ...... 1 Aspergillusflavus Infection ...... 1 Ear Moisture Loss Rate ...... 4

LITERATURE REVIEW AspergillusJlnvus, Its Infection, and Aflatoxin Production on Maize ...... 7 Aspergillus JJavus and Aflatoxins ...... 7 Colonization and Kernel Infection by A, flavus on Maize ...... 8 Growth of and Aflatoxin Production by A, fla v u s...... 14 Aflatoxin Detection and Kernel Infection Test ...... 19 Inoculation Techniques...... 23 Control of Infection by A, flavus and Aflatoxin Production ...... 26 Genetics of the Infection by A. JJavus and Aflatoxin Production ...... 35 Ear Moisture Loss Rate in Maize ...... 38 Ear Development and Ear Moisture Loss Rate in Maize ...... 38 Weather Factors Influencing Ear Moisture Loss Rate in Maize ...... 39 Agronomic Traits Related to Ear Moisture Loss Rate in Maize ...... 40 Genetics of Ear Moisture Loss Rate in Maize ...... 44 References ...... 46

CHAPTER I. A DIALLEL ANALYSIS FOR PERCENT KERNEL INFECTION BY ASPERGILLUS FLAVUS IN MAIZE Introduction ...... 62 Materials and Methods ...... 65 Results and Discussion ...... 67 References ...... 75

CHAPTER 2. A DIALLEL ANALYSIS OF EAR MOISTURE LOSS RATE IN MAIZE Introduction ...... 79 Materials and Methods ...... 81 Results and Discussion ...... 83 References ...... 94

iii GENERAL CONCLUSIONS

VITA ...... LIST OF TABLES Table 1.1 Mean percent kernel infection by Aspergillus flavus for 90 F, crosses, and general combining ability ( GCA ) effects and common parent means for 10 inbred lines. F, crosses were evaluated at Baton Rouge, LA in1992 and 1994 ...... 70

Table 1.2 Analysis of variance for percent kernel infection (PKI) by Aspergillus flavus for 90 F, crosses of maize evaluated at Baton Rouge, LA in 1992 and 1994 ...... 72

Table 1.3 F, crosses with significant specific combining ability (SCA) and / or significant reciprocal effects. F, hybrids were evaluated at Baton Rouge, LA in 1992 and1994 ...... 73

Table 2.1 Adjusted F, crosses means and line means for first probe moisture (30DPM)(g O.lg k g 1) and second probe moisture (51DPM) (g 0.1kg ') and ear moisture loss rate (EMLR) {g 0.1kg1 d ') for a 10-parent maize dial lei cross grown at Baton Rouge. LA in 1992 and 1994 ...... 88

Table 2.2 Analyses of variance for ear moisture loss rate (EMLR), first probe moisture (30DPM), and second probe moisture (51DPM) in maize grown at Baton Rouge, LA in 1992 and 1994 ...... 91

Table 2.3 General combining ability (GCA) effects for first probe moisture (30DPM), second probe moisture (51DPM), ear moisture loss rate (EMLR) from a 10-parent maize diallel cross grown in Baton Rouge. LA in 1992 and 1994 ...... 92

Table 2.4 Contrast sum of squares of first probe moisture (30DPM) (g 0.1kg ')2, second probe moisture (5IDPM)(g 0.1kg ‘)\ and ear moisture loss rate (EMLR)(g 0.1 kg 1 d 1)2 between L lines and out-of-state lines ...... 93

v ABSTRACT

Preharvest infection by Aspergillus JJavus Link ex Fries and subsequent aflatoxin contamination of maize (Zen mays L.) grain are major production problems. Ear moisture loss rate (EMLR) is an important component of hybrid performance and maturity, and to some extent, determines the suitability of a hybrid for a geographical area. A 10-parent diallel experiment was conducted in 1992 and 1994 to study the genetic nature of both percent kernel infection

(PK1) by A. JJavus and EMLR. General combining ability (GCA, Vj,), specific combining ability (SCA. V j, and reciprocal mean squares for PKI were significant. The ratio 2Vi, / ( +VJ was 0.54, indicating that both GCA and

SCA were important. The GCA. SCA, and reciprocal effects for PKI varied across years, which94 ' , ''cd that more reliable information could be obtained by conducting multi-year experiments.

The EMLR was measured with an electronic probe device. First probe moisture (30DPM) and second probe moisture (5IDPM) were recorded in the field at 30 and 51 days after midsilk, respectively. The GCA and reciprocal mean squares were significant, whereas SCA was not significant for EMLR. The 2Vy

/(2V,, + V j ratio was 0.81, indicating that GCA effects were more important than SCA effects for EMLR. LI08, L605. and L654 inbred lines showed significant, positive GCA effects for EMLR and Mol7 inbred line showed a negati\e GCA effect. 3UDPM and 5IDPM mean squares 1'or F, crosses were significant. A relatively low 30DPM, high EMLR, and positive GCA and

reciprocal effects for EMLR were important in lowering 5IDPM. Any selection scheme for lowering 51DPM or harvest grain moisture should exploit the extranuclear genetic component(s) for EMLR and use parental lines with significant, positive GCA effects for EMLR, and relatively low 30DPM. A significant correlation between PKI and EMLR was not delected in this study.

However, a significant genotypic correlation was found between PKI and

51DPM, indicating that the breeding goal of lowering both 51DPM and PKI by

A. JJavus can be achieved simultaneously. INTRODUCTION

Aspergillus flavus Infection

Since Lancaster et al. (1962) revealed that aflatoxin was the cause of the death of 100,000 turkey poultry on 500 farms in England in 1960, infection by

Aspergillus species and aflatoxin contamination have been extensively studied

(Lillehoj, 1987). Three main factors are responsible for stimulating these studies

(Hamilton 1987); aflatoxin causes myriad effects and economic losses in farm animals, aflatoxin is extremely carcinogenic in animals and may adversely affect human health. aHatoxin occurs in leedstuffs and foodstuffs, reducing the value of many agricultural products.

Kernel infection by A. flavus Link ex Fries and subsequent preharvest aflatoxin contamination of mai/e (Zea_jnaxs L.) kernels are two major and chronic production problems not only in the southeastern United States, but also in other parts of the world (Nichols, 1983; Zuber and Darrah, 1987; Kang et al.,

1988). The effects of aflatoxin on farm animals include malabsorption of various nutrients, decreased growth rates, decreased tissue integrity, reduced efficiency of feed conversion, reduced efficiency of reproduction, enhanced susceptibility to disease (Brown el al.. 1981; Richard et al., 1983; Cheeke and Shull, 1985;

Pier, 1987). The most serious consequence of Aflatoxicosis in animals is mortality (Lancaster et al.. 1962). Aflatoxin is a carcinogen in animals and may be a cause of human liver cancer (Boditie and Mortens. 1983; Pier, 1987). Aflatoxin contamination on major agricultural crops, such as maize (Zea mays L.), peanuts (A ra dits hypogaea L.) , rapeseed (Brassica napus L.), and cotton ( Gossypium hirsufum L.) seeds, can reduce grain quality and lead to an increase in the amount of nonmarketable grain, thereby causing a significant economic loss for farmers (Nichols, 1983; Wilson, 1987). Because of the reduced yield, devalued products, and noil-marketable grains due to aflatoxin contamination, farmers may suffer a large economic loss. In 1980, only maize growers in the southeastern United Stales lost about $97 million (Nichols, 1983).

Insecticides, cultural practices, garlic treatment, and competitive fungi have been employed to prevent or reduced infection by Aspergillus spp. and aflatoxin contamination ( Diener. 197b; Zuber and Lillchoj. 1979; Jones, 1987;

Barry. 1987; Kang, et al.. 1994). These techniques may reduce kernel infection rates and aflatoxin levels, but none of them can eliminate the problem. In the last two decades, many detoxification techniques have been developed to remove aflatoxin from aflatoxin contaminated food or feed (Lillchoj and Wall, 1987).

Although some of the methods can effectively remove aflatoxin to an acceptable level (20 ppb)(Lillehqj and Wall, 1987), they concurrently remove some desirable nutrients and require significant caplical investments, increasing the production cost of foodstuffs or feed stuffs (Lillchoj and Wall, 1987)

Aspergillus flavus is a special fungus known as facultative necrotroph. It not only can live on decaying plant parts but also can live on living tissues (Lillehoj, 1987). The initial source of primary inoculum in maize fields has been

thought to be conidia produced by mycelia grown from sclerotia that overwinter

in plant debris and litter in the soil (Jones, 1979). Conidia are either dispersed

by wind or carried off by insect vectors to maize ears (Wicklow, 1983). Growth

of conidia is dependent on conditions of the silk growth, temperature, humidity,

and other factors (Marsh and Payne, 1981;Davis and Diener, 1983). A. flavus

can colonize maize silks, glume tissue, and kernels without any damage to husks

(Jones et al.. 1980: Payne. 1983), Wounded kernels facilitate the invasion of A,

JJavus (Wallin. 198b).

The method for determining percent kernel infection (PKI) was developed

and modified by researchers (Adams et al. 1984; Tucker et al., 1986; Scott and

Zummo, 1988; 1990a; Scott et al.. 1991;). A significant and positive correlation

between PKI and aflatoxin concentration in maize kernels was found by Tucker

el al. (1986). Hybrids that show resistance to PKI have significantly lower

concentrations of aflatoxin in the grain at harvest (Scott et al., 1991).

Since the insecticides, cultural practices, anil detoxification techniques are either ineffective or not practical, developing resistant varieties may be the most effective and practical way of controlling infection by A. flavus and aflatoxin contamination. Efforts have been made to evaluate numerous germplasm (Zuber et al.. 1978; Lillehoj et al., 1983a; Kang et al.. 1988; Scott and Zummo, 1988;

Gorman et al.. 1992). Thus far, however, no maize genotype has been found to be completely resistant. It seems that more diversified germplasms need to be

screened in the future.

Although, the genetics of resistance to aflatoxin contamination has been

studied, the information obtained from these researches is not in agreement with

each other because of a major influence of environment on the infection and

aflatoxin production in different years and locations, and a lack of a uniform and

adequate inoculation technique (Zuber et al., 1978; Widslrom et al., 1984; 1987;

Darrah el al.. 1987; Kang el al.. 1991); Gorman et al.. 1992). Some progress has

been made in understanding the mechanisms of resistance to aflatoxin

contamination (Zuber et al., 1978; Widslrom et al.. 1984; 1987; Darrah et al.,

1987; Gorman et al.. 1992), but information on genetics of resistance to kernel

infection by A. JJavus is not available. Such information is important for plant

breeders who need to design a breeding program aimed at enhancing maize

resistance to infection by A. JJavus and aflatoxin production.

Ear Moisture Loss Kate

Lower harvest grain moisture in maize is an important breeding objective

to reduce growers' production cost associated with artificial grain drying and economic losses associated with delayed harvesting, such as lodging, dropped ears, bird damage, and diseases due to leaving maize in the field longer for

natural dry-down (Purdy and Crane. 1967a; Nass and Crane, 1970; Kang et al.,

1978). Ear moisture loss rate (EMLR) is closely related to harvest grain moisture content of maize (Kang et al., 1978; Cross. 1985; Cross et al. 1987; Kondapi et

al., 1993). Maize hybrids with rapid EMLR are especially needed in maize

production areas with a humid climate during both the grain maturation period

and the grain dry-down period following physiological maturity.

Ear drying in the Held is affected by temperature, humidity, and other

factors (Schmidt and Hallauer, 1966; Aldrich et al.. 1975; Kang et al., 1983,

1986). Significant phenotypic and/or genotypic correlations between EMLR and

other agronomic traits of maize, such as husk tightness, husk weight and number,

cob weight and length, kernel row number and depth, and pericarp permeability,

have been reported (Purdy and Crane. 1967b; Troyer and Ambrose, 1971; Kang et al.. 1975; Hicks et al.. 1976; Cavalieri and Smith. 1985).

Kang et al. (1978) developed a reliable, non-destructive method of

measuring EMLR in the Held. Practical uses of this electronic probe (DC-10

moisture meter) have been reported (Kang and Zuber. 1987, 1989; Kondapi et al.. 1993). Freppon et al. (1992) also reported the use of another portable electronic meter. JCS-l. for estimating ear moisture of maize in the field. These studies showed that correlation between ear moisture content (oven method) and probe moisture readings was high (r: = 0.82 to 0.85) and indicated that both

meters were reliable

There have been only a few genetic studies on EMLR in maize.

Researchers observed significant differences among inbred lines and hybrids under laboratory conditions and indicated that EMLR was a heritable trait (Crane et al., 1959; Hillson and Penny, 1965; Purdy and Crane, 1967a; Nass and Crane,

1970; Cross. 1985). Purdy and Crane (1967a) and Cross and Kabir (1989) conducted diallel studies on ear drying rates and reported that both GCA and

SCA effects were significant, with the additive genetic component being more important than the non-additive genetic component. Cross (1985) and Cross et al.

(1987, 1989) found that selection of inbred lines based on low ear moisture at 45 d post-pollination could effectively reduce ear moisture at harvest. Kondapi et al.

(1993) reported narrow-sense heritabilities for EMLR in two populations to be high, indicating a recurrent selection procedure should be effective in concentrating genes for this trait. There is little information available on EMLR, especially for the southern United States germplasms. during grain filling period and on relationship between EMLR and PKI.

The objectives of this investigation were to: I) determine GCA, SCA, and reciprocal effects for PKI in a 10-parent diallel cross; 2) determine GCA, SCA, and reciprocal effects for EMLR estimated in the field with the electronic probe device of Kang el al. (1978) in a 10-parent diallel cross and to identify inbred lines that could be used in a breeding program to lower harvest moisture of maize; and 3) delect any relationship between PKI and EMLR. LITERATURE REVIEW

Aspergillus flavus, Its Infection, and Aflatoxin Production on Maize

Aspergillus flavus and Aflatoxins

Aspergillus flavus Link ex Fries is one of the three fungi that produce aflatoxins that affect human health (Diener and Davis, 1987; Bodine and

Mertens, 1983; Pier, 1987; Wilson and Widstrom, 1995). Aspergillus flavus is a seed-inhabiting fungus that can contaminate a wide variety of crops before and after harvest- during handling, in storage, and during processing (Diener and

Davis, 1987). It can cause preharvest aflatoxin contamination naturally on most of the major crops such as, maize, peanuts, cottonseed, rice { Oryza sativa L.), and grain sorghum (Sorghum bicolor L.).

A . flavus is usually saprophytic (deriving nutrients from nonliving material), but it can also function as a parasite (deriving nutrients from living material) (Lillehoj. 1987). A. flavus reproduces asexually. Mycelium of the fungus can form conidia directly (Diener and Davis, 1987). Conidia are produced from conidiophores that are usually between 400 to 1000 pm in length and have roughened exterior walls (Wicklow, 1983). Conidia are dispersed either by wind or by insect vectors (Wicklow, 1983). According to Diener and Davis (1987), when conidia meet suitable conditions after they land on crops, they germinate and form a large amount of mycelium. Conidiophores are then formed from the mycelium. More conidia will be produced from the conidiophores and delivered 8

to other plants by wind or insects, which causes reinfection of A. flavus in the

field. Sclerotia are the principal overwintering form for A. JJavus. Sclerotium

forms naturally in standing maize and can be dispersed into field soils during

combine harvesting (Diener and Davis, 1987).

Aflatoxins are secondary metabolites of A. JJavus produced through the

polyketide biosynthetic pathway and are not necessary for the survival of A.

JJavus (Steyn and Vleggaar, 1986). Isolates or strains of A. JJavus vary widely in

their ability to produce aflatoxins. For example, A. JJavus usually produces two

kinds of allatoxins, BI and B2. But in Texas, 25-30% of 284 isolates of A. JJavus

from rice produced aflatoxins Bl and Gl (Diener, 1976). The aflatoxins can bind

to DNA, change the RNA structures copied from the DNA and may also prohibit

the transcription of DNA (Pier. 1987). Allatoxins have considerable effects on

livestock, such as reduced efficiency of feed conversion, decreased growth rate, enhanced susceptibility to diseases, decreased milk yield, reproductive problems, and increased sensitivity to extremes. (Bodine and Mertens, 1983; Hamilton,

1987). They also are carcinogenic to many animal species including rats, dogs, turkeys, pigs, rainbow trout, and may cause liver cancer in humans (Irvin, 1987).

Colonization and Kernel Infection o f A. JJavus on Maize

Colonization of A. JJavus on maize is dependent on the viability of inoculum, temperature, humidity, silk growth conditions, and other factors

(Jones, et al., 1981; Marsh and Payne. 1981; Davis and Diener, 1983; Jones, 1987; ). For a long time, infection by A. flavus and aflatoxin contamination of maize have been considered to be only a post-harvest problem (Shotwell, et al.,

1969). However. Lillehoj et al. (1975) indicated that aflatoxin contamination could also occur prior to harvest. Kernel Infection by A. flavus is an important but controversial topic. Infection by A. flavus and subsequent aflatoxin contamination has been recognized as serious preharvest problems since the mid-

1970’s (Zuber et al.. 1976; Lillehoj et al.. 1980b; Kang, et al., 1988).

The primary source of A. JJavus inoculum in maize Helds is not clearly known. One possible source may be airborne conidia (Jones. 1979; Jones et al.,

1981; Holtmeyer and Wallin. 1980). Holtmeyer and Wailin (1980) reported that in at least 17% of the days in a year, airborne conidia were trapped; Jones et al.

(1981) compared irrigated plots with unirrigaied plots and found that levels of airborne conidia in the unirrigated plots were higher than in irrigated plots and that these higher levels of airborne conidia correlated well with increased kernel infection by A. JJavus.

Where do the airborne conidia come from? Since airborne conidia can be dispersed by high altitude wind currents or rise from ventilating dusts on farm buildings and grain storage bins, Jones et al. (1979) postulated that storage areas and grain bins could be the sources of conidia. Conidia could also be produced from the sclerotium that overwintered in plant debris and litter in the soil (Jones et al. 1979). Sclerotia are naturally formed in standing maize and can be dispersed into field soils during combine harvesting. Thus, sclerotia may be another important source of primary inoculum of A. JJavus in maize (Diener and

Davis. 1987).

To determine whether A. flavus can be seed-borne, Kelly and Wallin

(1987) conducted an experiment originally designed for determining the best inoculation technique. They planted hybrid maize seed in the soil and then saturated it with an A. flavus conidia suspension at 2xl()'4 conidia ml'1. The treated and untreated control plants were transplanted into sterilized soil in the greenhouse. They took tissue samples from different growth stages for testing the presence of A. flavus on Aspergillus Differential Media (ADM). Surprisingly, they observed a systemic growth of A. JJavus in maize plant tissues. The fungus was recovered from leaves, stems, and roots at different growth stages. As the plant matured, the incidence and distribution of A, flavus was suppressed. Thus, whether or not contaminated seeds are another primary source of the fungus needs more experimental evidence.

Temperature and humidity are among the most important factors influencing the colonization and kernel infection by A. JJavus on maize. They both A9C affect the viability of A. JJavus spores (Fortnum, 1987). Teitell

(1958) reported that a relative humidity of approximately 75% at 29 C had a lethal effect on the conidia of A. JJavus. For stored maize grain, moisture levels of above 16% are favorable for invasion and colonization by A. flavus (Sauer and Tuite, 1987). Taubenhaus (1920) observed that erect maize ears in the field had

the greatest infection since erect maize ears might catch more water during

rainfall than droopy ears. Jones et al. (1980) reported that warm temperatures

(32 to 38 C) favored infection of kernels more than cool temperatures (21 to 26

C). Payne et al. (1983) showed that a temperature shift of 34/30 C in a day/night

caused a higher infection than a 26/22 C shift. It seems that in a certain range,

high temperature facilitates rapid growth of A. JJavus and gives it a competitive

advantage over other ear-inhabiting fungi (Fortnum, 1987).

Plant and silk growth have a significant effect on colonization and kernel

infection by A. flavus on maize, A. flavus can readily colonize both attached and

unattached silks (Jones et al. 1980: Marsh and Payne, 1984). After colonization,

A. flavus usually grows on the external silks, moves down the silks, and infects

maize kernels. Silk condition greatly intluenccs the processes of colonization and

kernel infection by A, flavus. Marsh and Payne (1984) reported that yellow-

brown silks gave the highest infection when the silks were inoculated with A. flavus spores. Brown silks also supported A. flavus growth (Payne, 1987), but

young green or dead silks were not favorable for colonization and kernel

infection by A. flavus. Jones el al. (1980) reported the highest infection when A. flavus spores were sprayed on silks two to three weeks after mid-silk. Aspergillus flavus can penetrate yellow-brown silks through both cracks and intercellular gaps

(Marsh and Payne. 1984). Colonization in silks is generally in the 12

parenchymatous tissue and growth of the fungus is parallel to the silk axis

(Payne. 1987). Colonization of A. JJavus also can happen in the kernels and the

cob pith. However, the colonization of A . JJavus in a particular kernel does not

require that the kernel be adjacent to a colonized silk (Marsh and Payne, 1984).

There is no consensus among researchers on the infection path of A. flavus

into maize kernels. Since silks were proven to be a main channel for A, flavus

colonization, many investigators believed that invasion by the fungus might

follow the same path as the pollen tube did (Anderson and Nehring, 1975; Payne,

1987). However, Marsh and Payne (1984) proved that the stylar canal was not

the major point of entry by A, flavus, whereas the tip cap region of a kernel was

the major entry point. Now many scientists believe that when the fungi reach the

kernel surface (by silks or other channels), they readily colonize the surface of

the kernels and the glume tissue surrounding the kernels. From these tissues, the

fungi can invade intact seeds through the junction of the bracts with their

rachillas and /or cob ( Payne. 1992). The internal infection by A. flavus on maize

kernels appears to occur late in the development of the kernel when kernel

moisture drops below 52% (Payne. 1987).

Some cultural practices also influence the colonization and kernel infection by A. flavus. Jones et al. (1981) observed a higher incidence of infection by A. flavus in a non-irrigated field than in an irrigated field. High plant density can

reduce the exposure of mai/e silks and ears to airborne fungal spores due to a 13

canopy effect (Jones, 1979), which, in turn, will reduce infection. Planting date

had an effect on infection by A. jlavus, but the effect was confounded by

environmental conditions (Jones, 1987). Thus, whether early or late planting is

better depends on the weather and insect incidence in a particular year and

location. Weed competition may cause more infection in maize fields (Jones,

1987). Increased inoculum levels also lead to greater infection (Payne, 1983).

Insects may be the most important factor influencing the colonization and

kernel infection by A, flavus. As early as 1920, a close association between A. flavus infection and insect injury in maize was reported (Taubenhaus, 1920).

Since aflatoxin contamination was recognized as a preharvest problem in the

1970s. insect damage was extensively reported to be related to infection by A.

Jlavus and aflatoxin contamination (Anderson el al.. 1973; Fennell et al., 1975;

Widslrom et al; 1975; McMillian, 1987; McMillian et al; 1978; 1985; 1991).

Lillehoj et al. (1982a) used three isolates of A. jlavus to inoculate maize ears and

then collected European Corn Borer (ECB) ( Osirinia nubilalis Hubner) larvae

from the inoculated ears. They found that A. jlavus isolates were recovered from

the inside of the larvae, indicating that ECB could be a natural vector of A. flavus. From a six year study in Georgia. McMillian et al. (1985) observed

significant, positive correlations between the percentage of ears with visible A. flavus and percentage of ears with visible insect damage (0.17**) and between the percentage of ears with visible A. jlavus and the amount of insect damage to 14

ears measured in centimeters of feeding penetration down the ear (0.50**).

Fennell et al. (1975) reported that a higher percent kernel infection by A, flavus

was observed when ears were damaged by insects (6.3%) compared to the ears

which were not damaged by insects (2.5%). Payne (1992) gave four possible

mechanisms by which insects may increase theA. Jlavus infection: 1) transport

primary inoculum to the ears, 2) move the inoculum already on the silk into the

kernel, 3) disseminate inoculum within the ear, and 4) predispose kernels to A.

jlavus invasion.

Many insects, such as the maize weevil (Sitophifus zeamais), ECB, fall

armyworm (FAW) (Spocbptera frugiperda), corn earworm (CEW) ( Heliothis

zea), can carry spores externally and/or internally (Widslrom, 1979). Results are

not consistent in ranking which insect species was the most effective vector in

terms of infection rales and allaloxin levels (Fennell et al.,1975; Widslrom et

al., 1975). Genotypes with resistance to insects should reduce infection by A. jlavus and subsequent aflatoxin accumulation. However, experimental evidence

indicated that genotypes with resistance or susceptibility to ECB and CEW

showed no significant differences in the amount of aflatoxin produced (Guthrie

et al., 1982).

Growth of and Aflatoxin Production by A. flavus

Once A. jlavus colonizes maize kernels, it grows and produces aflatoxin.

Marsh and Payne (1984) described the process of A. jlavus growth after it went into yellow-brown silks as follows: after 4 to 8 hours, conidia of A. Jlavus germinated on inoculated silks and then mycelium grew and spread rapidly across the silk; by 48 hours, conidiophores and conidia were formed. Aflatoxin is produced rapidly after the colonozation by A. Jlavus. In a pinboard-inoculated experiment, Thompson et al. (1983) found that after only two weeks aflatoxin reached 238 ppb in the inoculated ears. Payne et al. (1988) reported that aflatoxin appeared within one week in wounded-inoculated ears and that aflatoxin concentration increased linearly until 7th to 9th weeks. McMillian et al. (1991) showed that hybrids had a unique linear rate of allaloxin increase during ear maturation. The linear increase of aflatoxin was also reported by other researchers (Widslrom et al.. 1986). The maximum aflatoxin accumulation usually appeared between 50 to 65 days post-silking (Lillehoj et al. 1975; Payne et al. 1988).

Factors affecting the growth of A. jlavus and aflatoxin production are substrate, isolates, moisture, temperature, aeration, etc (Lillehoj, 1983). Growth of A Jlavus and aflatoxin production have been observed on most grain crops that can serve as natural substrates (Lillehoj, 1983). Diener (1976) has shown that sucrose, glucose, fructose, glycerol, ribose, and xylose are the best carbon sources for aflatoxin formation; glycine and glutamate were the best single amino acid nitrogen sources: that zinc is critical for maximal aflatoxin production; and that iron and magnesium are also essential for toxin production by A. jlavus. 16

Isolates of A. Jlavus are quite different in their aflatoxin production

capabilities on different or same substrates. Diener (1976) reported that isolate

213 of A. Jlavus from Texas produced 0 to 349 jug g 1 aflatoxin on peanuts,

whereas 0 to 238^g g 1 was produced on rough rice; isolate NRRL 3145

produced 50-900% less aflatoxin than isolates NRRL 3000 and NRRL 2999 did.

Diener and Davis (1987) compared seven A. Jlavus isolates for aflatoxin

production on 15-20% sucrose medium. They found that the amount of aflatoxin

produced by different isolates was different.

Temperature and moisture are the two most important factors influencing

the growth of/I. Jlavus. These two factors usually interact strongly. The fungus

grows best at 36 to 38 C. with a range of 6 to 46 C. but it may grow at higher

temperatures on field maize (Lillehoj. 1987). The optimal temperature for

aflatoxin production by A. Jlavus is 24-25 C. Sisson (1987) reported that the

severity of aflatoxin contamination was related to July average maximum

temperature. August average minimum temperature, and July average high

humidity. Diener and Davis (1966) reported maximum aflatoxin production by

A. Jlavus at 25 C on peanuts. However. Diener (1973) found that the optimal

temperature for the production of aflatoxin B1 was 30 to 35 C for II days,

whereas for Gl it was 20 to 25 C for 9-11 days. Zuber et al. (1983) noted that

high temperatures during the growing season coincided with high aflatoxin levels

in open-pollinated maize varieties and hybrids. 17

The lowest moisture content

development on a starchy grain was 17.5% (Lopez and Christensen, 1967). Sauer

and Tuite (1987) found that when relative humidity (RH) increased above 85%

(which is equal to 16.7% MC in grain), the growth of A. JJavus increased

dramatically; in this range even a small increase in RH could increase the

aflatoxin contamination significantly. In most situations, the MC of the grain

rather than RH is measured. Since there are many factors influencing MC of

grain, we cannot set an absolute safe MC level for maize storage. However,

Sauer and Tuite (1987) indicated that at an MC of 16% or less, growth of A.

JJavus was restricted, therefore 17% MC might be low enough to slow growth

of A. JJavus. Humidity also influences production of aflatoxin. Sauer and Tuite

(1987) found that when RH was higher than 85% , production of aflatoxin B1

increased dramatically. Lillehoj (1983) reported that A. JJavus growth and

aflatoxin synthesis were minimal below 85%’ RH but that quite a bit of aflatoxin

was detected at 86-87% RH.

A. JJavus is an aerobic organism and its growth is highly affected by

changes in atmospheric gas composition. CO: and 0 3 are the most important

gases that influence the growth of A. JJavus and subsequent aflatoxin production.

Sanders et al. (1968) showed that increased CO: levels decreased aflatoxin

production. Diener and Davis (1969) reported that growth and aflatoxin

production by A. JJavus in peanut kernels were reduced when C 03 increased from 18

20 to 100%; at 100% CO, there was no growth or aflatoxin production. Landers et al. (1967) also showed that increasing C 0 2 levels above 20% decreased fungal growth and aflatoxin production; 0 2 levels below 5% also reduced fungal growth and aflatoxin production. Diener and Davis (1969) also reported that when 0 2

levels were reduced from 5 to 1%, aflatoxin production was decreaded. These

studies showed a possible way of controlling aflatoxin production by A. favus in a storage grain bin.

In the Held, aflatoxin production is often associated with insect incidence,

plant resistance, temperature, water stress, and nitrogen stress. McMillian (1987)

reported that insect damage was closely related to aflatoxin production level. A three year field study at Tilton showed that all ears damaged by major maize insects had a higher levels of aflatoxin than those of an undamaged check. Maize differences in resistance to infection by A. JJavus and aflatoxin production have been clearly demonstrated for hybrids and inbreds (Lillehoj el al., 1976; LaPrade and Manwiller, 1976; Widstrom et al. 1984). Lillehoj et al. (1976) found that significant differences in aflatoxin Bl levels among six hybrids. Widstrom et al.

(1984) compared aflatoxin contamination levels of dent maize and sweetcorn inbreds; significant differences in aflatoxin levels were detected among both dent maize inbreds and sweetcorn inbreds.

A Held survey in Georgia indicated that high aflatoxin levels in maize were associated with drought stress in 1977 in the southeastern USA (McMillian et al.. 19

1978). Irrigation can reduce aflatoxin production to a level similar to that found under adequate natural rainfall. Fortnum and Manwiller (1985) observed that irrigation in a maize field suppressed aflatoxin production in 14 hybrids grown in South Carolina in 1979. McMillian et al. (1991) in summarizing data from a

13-year field survey found that high temperature, high humidity, and heavy morning dew during ear development favored aflatoxin development; aflatoxin contamination was positively correlated with mean temperature in the field

{0.86*); and that irrigated cornfields in Georgia usually sustained significantly less aflatoxin contamination than non-irrigated fields. In addition to temperature and water stress, nitrogen stress also has been linked to high aflatoxin levels in maize. Payne et al. (1989) reported that when no nitrogen fertilization was applied to plants, ears contained, on average. 28% more aflatoxin than the ears from the plants receiving optimal nitrogen fertilization.

Aflatoxin Detection and Kernel Infection Test

Aflatoxin detection and kernel infection test techniques are bases for the research for screening genotypes with resistance to kernel infection by A. Jlavus and aflatoxin production. A bright greenish-yellow fluorescence (BGYF) under longwave (365 mu) ultraviolet is an indicator of the presence of aflatoxin in maize (Fennell et al. 1973). The BGYF technique was once used as a rapid screening method to detect aflatoxin-contaminaled lots at the time of marketing.

Although this test is not always accurate and is not an absolute measure of the 20 quantitative amount of aflatoxin, it is a good indicator of the presence of aflatoxin. Dickens et al. (1980) found that the BGYF kernels from a 1134-kg sample contained an average of 8665 kg 1 aflatoxin. whereas the non-BGYF sample had an average of 46 fig kg'1. Lillehoj et al. (1982b) studied 30 hybrids from the 1979 South Carolina yield trials to examine the relationship between

BGYF and aflatoxin contamination; a significant association between the number of BGYF-tluorescing particles and the aflatoxin concentration was found.

Shotwell (1983) reported that mai/e samples with no BGYF particles had a very low probability of containing aflatoxin. Lillehoj et al. (1983) reported a positive and significant correlation between aflatoxin and BGYF, Since BGYF is only an indicator of the presence of aflatoxin. Wilson (1987) suggested that it can only be used to identify lots for further chemical analyses and should never be used to set mai/e prices in the market place. Another rapid screening technique for aflatoxin uaes the minicolumn. Several minicolumn techniques used for detecting aflatoxin were reviewed by Wilson (1987). But these minicolumn techniques, like

BGYF. cannot be used for quantitative purposes.

More accurate quantitative procedures, such as thin layer chromatography

(TLC) and high pressure liquid chromatography (HPLC), have been developed

(Davis and Diener, 1980; Shotwell, 1986; Wilson, 1987). The Association of

Official Analytical Chemists (AOAC) (1984) adopted and recommended the

Contaminant Branch (CB) method for aflatoxin analyses in maize. The CB 21

method is an excellent TLC method and is also the standard by which other

methods are judged. The HPLC method for separating and detecting aflatoxins

is similar to TLC but HPLC is much faster than TLC. The major steps involved

are aflatoxin extraction, purification, and quantification. Two other techniques

recently used to detect aflatoxin Bl are solid phase radio-immunoassay (RIA) and

enzyme-linked immunosorbent assay (ELISA) (El-NaKib et al., 1981). The

ELISA is a qualitative and/or semi-quantitative method and is more suitable for

field use than RIA method (Wilson, 1987). In 1994. Keller et al. (1994) found

that norsolorinic acid (NOR), a visible orange intermediate of aflatoxin, can be

seen in specific maize kernel tissues (embryo and aleurone) and specific fungal

tissues: thus, the appearance of NOR indicated kernel infection by A. JJavus,

However, application of this technique has not been reported yet.

Kernel infection lest procedures have been developed and used by many

researchers (Adams et al.. 1984; Tucker et al.. 1986; Scott and Zummo,1988;

Scott et al.. 1991). Adams et al. (1984) studied laboratory techniques that could be used to detect differences in responses to A. JJavus growth among maize genotypes. They found that kernel infection rates among genotypes were significantly different regardless of the developmental stages of ears. A procedure

used for determining percent kernel infection (PKI) was described by Scott et al.(1991 j as follows: Kernels to be assayed for fungal infection were randomly selected from whole kernels of all inoculated ears. Then the kernels were surface- 22 sterilized by dipping them for 1 min in 70 mL/L aqueous ethanol, soaked for 3 min in 1.25% NaOCl (laundry bleach), and rinsed in sterile, distilled water.

Three hundred and ninety kernels per treatment were plated in 100-mm petri dishes (13 kernels per dish) on Czapek solution agar amended with 7.5% sodium chloride to restrict growth of other fungi and bacteria. After 6 d of incubation at

28 ± 2" C, plates were examined for A. fla vu sgrowth (green-yellowish spores appear) and percent kernels infected with A. fla vu s was computed. This procedure has been used by other researchers (Tucker et al., 1986; Zummo and

Scott. 1989) for screening genotypes.

To get an accurate estimate of aflatoxin concentration in maize kernels, an appropriate sampling technique should be used since aflatoxin concentration in individual kernels of a lot varies widely. Lee et al. (1980) reported that toxin concentrations of individual kernels from a maize lot ranged from 0 to 500,000 jug kg V Several sampling methods, such as stream sampling, probe sampling, and field sampling, were recommended by Davis et al. (1980) to insure that sample concentration was in reasonable agreement with the lot concentration.

Kernel infection by A. flavus and subsequent aflatoxin production are a continuous process during and alter kernel development, therefore, kernel infection must be related to aflatoxin concentration in A. /tow/s-contaminatcd maize kernels. Tucker et al. (1986) first found that PKI was significantly correlated with total aflatoxin concentration and suggested that PKI would be a 23

valid predictive estimate of aflatoxin concentration in maize. Payne et al. (1989)

used a spray silk inoculation method to study the relationship between PKI and aflatoxin contamination; a significant correlation (0.91*) between PKI and aflatoxin levels was detected in A. ./famy-inoculated ears. Scott et al. (1991) reported that the hybrids previously classified as resistant to kernel infection by

A. Jlavus had fewer kernels infected and lower aflatoxin concentration in the corn grain at harvest.

Inoculation Techniques

The first and most important step in developing a resistant variety is to identify genotypes having genes for resistance. Since the infection by A. Jlavus and subsequent aflatoxin production vary widely with different environments, plant growing conditions, and insect incidence (Rambo et al.. 1974; Lillehoj et al., 1975; Fennell et al., 1975; Anderson et al.. 1975; Zuber et al., 1979; Jones et al.. 1980). idcntilicalion of resistant genotypes under natural infection by A.

Jlavus is not reliable enough for screening for resistance.

Since 1970s, efforts have been made to develop artificial inoculation techniques. Rambo et al. (1974) designed three inoculation methods to evaluate four hybrids for incidence of visible A. jlavus infection and BGYF. The methods were I) silk spray: spraying silks with a spore suspension; 2) needle injury: injecting spore suspension into ears with a syringe and needle through the husk; and 3) cob hole: inserting a swab dusted with spores into a hole drilled into the 24

cob. They did not 11 nd any visible infection from silk spray. However, visible

kernel infection was observed with the needle-injury and cob-hole methods.

These results indicated that kernel injury might be necessary for infection by A.

Jlavus to begin. For evaluating infection incidences of five single-cross hybrids

and one short-season cultivar, LaPrade and Manwiller (1976) used four

inoculation methods; they were:l) silk inoculation: spores sprayed on silks; 2)

kernel surface inoculation: spores sprayed on the surface of uninjured kernels of

each ear; 3) single kernel inside inoculation: spores injected into a single kernel,

and 4) three kernel inside inoculation: spores injected into three kernels per ear.

Significant differences in kernel infection and aflatoxin production were found in

both kernel inside inoculation methods. These results also suggested that

wounding of kernel seemed to be prerequisite for kernel infection by A. Jlavus.

More injury inoculation techniques were developed by Calvert et al. (1978). They

designed three kernel injury methods: I) pinboard method: puncture holes in

kernels with a pinboard (sewing pins arranged in rows and mounted on a holder);

2) razor blade method: slit kernel surfaces by razor blades mounted in a holder;

and 3) needle injury method: make holes in kernels by.a hypodermic syringe and

needle. After holes or slits were made, inoculum was applied. Higher aflatoxin

levels were found in both thick and thin pericarp genotypes when pinboard and

razor blade methods were used. This result further indicated the importance of

kernel injury for effective inoculation with A. Jlavus in maize. 25

Kernel injury techniques, however, may circumvent any resistance

associated with integuments of maize kernel to natural infection by A. flavus.

Wallin (1986) compared aflatoxin production between whole kernels and

decappcd kernels. He found that decapped kernels produced more aflatoxin than

intact kernels, suggesting that the kernel integuments, i.e., pericarp and aleurone

layers might contribute to resistance. To accurately evaluate resistance of maize genotypes to natural infection by A. jla vu s. non-injury techniques seemed to be

more suitable. Tucker et al. (1986) compared two non-injury inoculation

techniques with two injury inoculation techniques. The two non-injury techniques

were I) silk atomizing: atomizing conidia onto silks and then the silks were covered with a plastic bag and a pollinating bag: 2) kernel exposure : husks were pulled from one side of an ear to expose about 25% of the ear surface and then conidia were atomi/cd onto the surface. The husks were then repositioned and the inoculated ear was covered with a pollinating bag. The two injury techniques were 1) pinboard method as described above and 2) knife method: the tip of a paring knife was dipped into a conidia suspension and then plunged through the husks into underlying kernels. They found that ail four methods could differentiate among hybrids in terms of PKI. But only the pinboard technique permitted a complete differentiation. Darrah et al. (1987), by using the atomizing silk method as described above (they called it modified natural inoculation), successfully differentiated hybrids for aflatoxin B1 production. 26

Zummo and Scott (1989) compared the pinboard method with five non-injury methods. The five non-injury methods were:l) needle inoculation into silk channel, 2) needle inoculation through husk, 3) toothpick in ear, 4) toothpick in silk channel, and 5) string on maize silk. They found that two non-injury techniques (the needle in silk channel and the needle through husk) produced approximately equal levels of kernel infection compared to pinboard method and separated resistant and susceptible maize genotypes. The needle in the silk channel was recommended for it was faster and easier to apply than the pinboard technique.

Recently, a special inoculator has been developed by Campbell and White

(1994). This device functions similar to the pinboard method. By evaluating commercial maize hybrids, they have shown that this inoculating device allows for more rapid evaluation of genotypes resistant to A. Jlavus infection and aflatoxin production.

Control of Infection by A. flavus and Aflatoxin Production

Since insect damage has been shown to be correlated with kernel infection by A. jla vu sand aflatoxin production, insecticides could be an effective way to control infection by A. jla vu sin the field. Insecticides will reduce insect vectors of A. jla v u s and. in turn, reduce the infection by A. jla vu s and subsequent aflatoxin contamination. Lillehoj et al. (1976a) reported that insecticide applied to silks of developing ears in the field generally reduced insect damage and 27

aflatoxin contamination, hut it could not completely eliminate the problem. This

is because insects are not the only vectors of A. jla vu s spores (Jones, 1979).

Fungicide applied to developing silks did not significantly reduce A. jla vu s

infection and aflatoxin production (Lillehoj et al., 1980c). Furthermore, the use

of insecticides or fungicides to control insects and aflatoxin contamination in

maize is not economical. However, when fungicides are applied to stored maize

grain, incidence of Aspergillus spp. can be significantly reduced ( White et al.,

1993; White and Toman, 1994; Toman and White. 1994).

Biological control methods have been effectively used for many diseases.

A biological control strategy can be to find a non-toxigenic strain of a fungus that can outcompete the toxigenic strains in the same ecological niche. Diener (1976)

found that A. niger could prevent the penetration of peanut pods by A. jla vu sand

marked antagonism between these two fungi was noted in petri plate cultures; several fungi were able to break down aflatoxin in peanuts. Therefore, he suggested that microbial competition or microbial breakdown could probably be employed to lower levels of aflatoxin. Brown et al. (1991) studied the inhibitory effect of an atoxigenie A. jlavus (strain 36) on aflatoxin production by a toxigenic

A. jla vu s (strain 13): they found that the atoxigenie strain significantly reduced preharvest aflatoxin contamination by the toxigenic A. jlavus. Zummo and Scott

(1992) compared kernel infection of maize ears inoculated with both Fusarium monilifornw and A. jla vu sand with A. jla vu salone: significantly fewer kernels 28

were infected with A. Jlavus in ears inoculated with hotli fungi than kernels from

ears inoculated with A. fla vu s alone, indicating that F. m oniliform ecan inhibit

kernel infection by A. fla vu s in inoculated maize ears, Doehlert et al. (1993)

found that exogenous lipase applied to soybean cotyledons resulted in the

generation of volatile aldehydes by the lipoxygenase pathway that halted the

growth of A. fla v u; s when lipase was added to the soybean homogenate, all

fungal spore germination was inhibited. They believed that hexanal, a product of

the lipoxygenase pathway with known antifungal activity, was the major volatile

substance generated from lipase-treated homogenales. With the development of

modern molecular genetics and biochemistry, attempts to isolate genes coding for

the enzymes in the aflatoxin synthesis pathway have been successful (Payne,

1992). Three ways of how this information could be used to control aflatoxin

contamination were postulated by Payne (1992): 1) to engineer a biological

control agent; 2) to identify compounds that block aflatoxin formation; and 3) to

identify compounds that inhibit the fungus. A better approach for making

potential biocontrol agents or noil-toxigenic strains is to use site-directed mutation

in toxic fungi at several base points using molecular techniques. The compounds

that either specifically inhibit the transcription of pathway genes or inhibit the pathway enzymes may be the best compounds we need to look for. A controlling

strategy could be to look for a compound that can inhibit or kill the aflatoxin- producing fungi and then engineer plants to produce the compound. 29

Garlic (Allium sativum L.) is known for its medicinal, antimicrobial, and

pesticidal properties. Aqueous extracts of garlic bulbs inhibit growth of many

species of fungi (Tansey and Appleton, 1975). Mabrouk and El-Shayeb (1981)

used minced garlic and aqueous garlic extracts to interfere with mycelial growth

of and aflatoxin formation by A. Jlavus. They found that both minced garlic and

aqueous garlic extracts were inhibitory to aflatoxin formation at lower

concentrations (0.1- 0.5 g 100ml ‘) and fungicidal at higher concentrations (5-10

g 100ml *). Kang et al. (1994) used aqueous extracts to determine the

concentrations that can effectively inhibit the growth of A. Jlavus and A.

parasiticus: when garlic concentration (1 g of garlic in 2 ml H;0 = 100%) was

5% or above, growth of both fungi was inhibited. Field research further showed

that a 50%' concentration of garlic extract sprayed on silks before applying A.

Jlavus significantly reduced the percent kernel infection, Kang et al. (1995) also

reported that the inhibition by a cultivated garlic extract was higher than that by

a wild garlic extract. Tadi et al, (1991) reported that two organosulfur

compounds of garlic (ajoene and diallyl sulfide) and crude garlic extract inhibited

mutagenesis induced by aflatoxin Bl; a significant decrease in organosoluble

metabolites of aflatoxin Bl was observed with ajoene and garlic extract. If the

ajoene is the compound Payne (1992) referred to. scientists may be able to

isolate and clone the gene that codes for this compound and engineer it into the 30 matze genome. Molecular genetics and biochemistry may offer new approaches to solve these problems.

In addition to garlic, other plant extracts also have been reported to have inhibitory effects. Bilgrami et al. (1992) found that the growth of A. Jlavus and aflatoxin production could be inhibited by eugenol and onion extracts as well as garlic extract. Sinlta et al. (1993) showed that 100 /xg ml 1 clove oil and 150 jug m l1 cinnamon oil significantly inhibited the growth of A. flavus and reduced aflatoxin production in liquid medium.

Detoxification is an immediate and effective approach. It has been extensively studied for removing allatoxins from many crops, such as maize, peanut, cottonseed, rape.seed, etc.. Researchers have focused on developing procedures for detoxifying target commodities to provide feed-grade materials, trying to avoid massive economic loss. Many techniques, such as physical separation of allatoxin-conlaminated seed, solvent extraction of aflatoxin, and chemical inactivation of the toxin, have been developed (Lillehoj and Wall,

1987).

In the peanut industry, kernels are traditionally examined at shelling to remove discolored and immature seed by hand sorting and/or mechanical screening (Goldblalt. 1971). In addition, electronic sorters have been developed to individually examine each kernel and reject discolored seed, resulting in a concomitant reduction of aflatoxin in the parent lots (Dickens and Whitaker, 31

1975). But a similar sorting technique has not been developed for maize since

maize kernel fragments are not suitable for electronic sorting (Lillehoj and Wall,

1987).

Many heat treatments have been employed in aflatoxin-detoxification studies (Rehana et al., 1979; Seenapa and Nyagahungu, 1982), such as steaming under pressure, dry roasting, and other cooking techniques (Coomes, et al.,

1966; 1983; 1985; Schroeder. et al.. 1985). These techniques often reduce aflatoxin levels but do not eliminate the toxin,

Ultraviolet radiation and gamma irradiation have been used to reduce aflatoxin levels in contaminated peanut cakes and vegetable oils (Shantha el al.,

1981). Ultraviolet light has been employed to degrade aflatoxin M, in milk

(Yousef and Marlh. 1985). The results from the use of gamma-ray treatments to remove aflatoxin are mixed. Feuell (1966) observed no reduction in toxicity, whereas Ogbadu and Bassir (1979) detected decreased toxicilies of contaminated feeds after gamma irradiation.

Chemical procedures, such as solvent extraction of aflatoxin from contaminated commodities, chemical inactivation of aflatoxin in situ, and oxidation and reduction agents for degradation and destruction of aflatoxin have been reported (Feuell, 1966; Goldblatt, 1971; Detroy el al. 1971). Several solvents have proven capable of removing aflatoxin from contaminated oilseed meal, peanut meal, and cottonseed (Feuell. 1966; Goldblatt. 1971). Recently, 32

Phillips (1995) reported that a phyllosilicate clay could tightly hind to aflatoxins and markedly decrease the bioavailability of radiolabeled allaloxins. One negative feature of solvent extractions is that it also removes some desired portions of the seed and reduces the net value of the commodity. Detroy et al. (1971) found that there were two highly vulnerable sites on the aflatoxin molecule for chemical reaction: the coumarin moiety and the double bond of the terminal furan. Thus, chemical inactivation of aflatoxin is a promising approach to detoxify commodities (Lillehoj and Wall. 1987). Many oxidation and reduction agents can effectively reduce aflatoxin contaminants in different crops (Goldblatt,

1971). However, they usually can only partially eliminate one or two kinds of allaloxins from commodities and in most cases will cause some loss of desirable components in the commodity.

Aqueous or gaseous ammonia with or without elevated temperature and pressure appears to be the most efficient approach to decontaminating commodities containing allatoxin (Lillehoj and Wall. 1987). Gardner et al. (1971) developed optimum techniques to decontaminate ton lots of oilseed meal by ammonification. Lillehoj and Wall (1987) described in detail an NH,-air procedure developed by Brekke et al. (1978) for decontaminating aflatoxin. The procedure is: recirculate NH,-air mixtures through a column of shelled maize at

25 C and 17.b% moisture until the NH, was uniformly distributed; after ammoniation. the columns are sealed and incubated at 25 C for two weeks. This procedure resulted in a reduction of initial aflatoxin levels from 100 ppb to 10 ppb. When this procedure was expanded to larger lots (about 40 tons), using a standard grain bin, it effectively reduced aflatoxin from levels exceeding 100 ppb to less than 20 ppb (Brekke, et al., 1978). The feed efficiency of ammonia- treated maize grain has not shown any negative effects (Brekke et al., 1978).

However, as pointed out by Lillehoj and Wall (1987), strong ammonia odor and discoloration of treated-maize kernels are still major problems that need to be solved and extensive feeding studies should be undertaken to define the fate of the ammonia degraded toxins.

The most economic and practical approach to control infection by A. Jlavus and aflatoxin production may be to develop resistant maize varieties. In order to incorporate resistance to infection by A. flavus and aflatoxin contamination into maize germplasm. a breeder must I) identify sources of resistance and 2) determine the genetic control of resistance (Gorman, 1992). Significant differences in aflatoxin contamination among maize genotypes have been reported by many researchers. LaPrade and Marnviller (1976) evaluated five single-cross hybrids and one short-season maize eultivar for aflatoxin production; significant differences among hybrids for aflatoxin concentration were found. Lillehoj et al.

(1976b) studied five South Carolina experimental single crosses and a commercial single cross hybrid. They observed significant differences among genotypes for

BGYF and aflatoxin concentration. Zuber et al. (1976) and Calvert et al. (1978) 34

reported that kernels with a thick pericarp had significantly higher levels of

aflatoxin did kernels with a thin ]>eriearp. Widstrom et al. (1978) investigated 20

commercial hybrids and 30 dent and 13 sweet corn three-way crosses and found

that the commercial hybrids showed significant differences for aflatoxin levels at

Tifton, but significant differences were not detected when aflatoxin concentrations

were averaged over all locations and no significant difference was detected in

three-way maize hybrids. Zuber el al. (1978) studied aflatoxin Bl production in an 8-parent diallei: significant differences in aflatoxin Bl levels were detected among both inbreds and hybrids. These studies and others convincingly demonstrate that inherent differences in resistance to aflatoxin production exist among commercial hybrids (Lillehoj et al.. 1976b: LaPrade and Manwiller, 1977;

Widstrom et al.. 1978; Widstrom et al., 1984; Kang et a!., 1988), among experimental single crosses (Zuber et al., 1978; Widstrom et al.. 1984; Scott and

Zuniino, 1991; Gorman et al.. 1991; Widstrom and Wilson, 1993; Zhang et al.,

1993), and among inbreds (Zuber et al., 1978; Scott and Zummo, 1988; 1990a;

Scott et al.. 1991), Recently, two maize inbred lines showing resistance to PKI and allatoxin production have been released (Scott and Zummo, 1990b; 1992).

Since infection by A. Jlavus and subsequent aflatoxin production are greatly influenced by many factors such as environments, inoculation techniques, insect incidence, and silk growth stages, etc.. the results of screening and identification of resistant maize lack repeatability and are difficult to compare 35 with each other. However, one point can be made: there are no known maize genotypes with complete resistance to infection by A. Jlavus and aflatoxin production. A great diversity of maize genotypes needs to be screened in the future.

Genetics of Infection by A. flavus and Aflatoxin Production

To design an effective breeding program aimed at enhancing maize resistance to kernel infection by A. flavus and aflatoxin production, breeders need genetic information about the pattern of gene action controlling this resistance.

Unfortunately, information on this aspect is limited and inconsistent. The genetics of resistance to kernel infection by A. fla vu sremains poorly understood.

Zuber et al. (1978) conducted the first genetic study on maize resistance to aflatoxin Bl production using an eight inbred diallel; they used A. fla vu s isolate NRRL 3357 and the pinboard inoculation technique. Bulked kernels from inoculated ear were used for aflatoxin Bl analysis. Results showed that the GCA was significant, whereas SCA and reciprocal mean squares were not; suggesting that the GCA effect, or the additive genetic component of variance was important in controlling aflatoxin resistance in maize. Based on this result, they suggested that a cyclic selection program would be effective for developing a maize population with resistance to aflatoxin contamination. Widstrom et al. (1984) used an eight-inbred diallel of sweet corn and a nine-inbred diallel of dent corn to evaluate aflatoxin resistance in maize; A. jlavus isolate NRRL 3357 and a knife 36 inoculation technique were used. They reported that when three-year data were used, significant GCA effects were detected among inbreds in both dent and sweet corn diallels. A significant SCA was not found in either diallel. Variations for GCA and SCA effects within years were noted. This research further showed that additive genetic effects might be the main genetic components responsible for aflatoxin resistance in maize. Gardner et al. (1987) used seven of the eight inbred lines used by Zuber et al. (1978) lo estimate allaloxin contamination; isolate

NRRL 3357 and the pinboard inoculation method were employed; but only the kernels showing visible damage by the pinboard were assayed for afiatoxins.

They observed that both GCA and SCA mean squares were significant. Darrah et al. (1987). noticing that damaged kernels might overcome any resistance that might exist in the pericarp and aleurone tissue of the kernel, studied the same seven inbreds as used by Gardner el al. (1987) by using a modified natural inoculation method. In this study, spores were sprayed on the silks and ears were covered with plastic and tassel bags; aflatoxin Bl levels were observed to be significantly different among genotypes; and the GCA mean square was significant, whereas the SCA mean square was not. These results were different from those obtained by Gardner el al. (1987), The authors argued that the inconsistency in the two studies was because of different inoculation methods employed. Gorman et al. (1992) studied GCA and SCA of resistance to aflatoxin accumulation in maize grain using a diallel cross made from seven Lfy synthetics 37 in three environments; isolate SRRC 2999 of A. parasiticus Speare and modified knife inoculation technique (dipping a knife into a conidia suspension, cutting through the husks, and then injuring one kernel row) were used. The results indicated nonsignificant genetic variation among the 21 F, crosses. There was a preponderance of environmental effects. This result is different from previous studies. The authors suggested that different genetic systems may govern the resistance, depending on the germplasm. environmental conditions, and inoculation procedure. Campbell and While (1995) recently studied the inheritance of resistance of maize to Aspergillus ear rot caused by A. Jlavus by investigating Ft. F;. F,. and backcross generations of 11 crosses between resistant and susceptible inbreds; they found that additive and dominance gene actions are of primary importance in resistance to Aspergillus ear rot.

A general conclusion from the genetic studies is that maize resistance to infection by A. jla vu s and subsequent aflatoxin contamination is genetically controlled in a quantitative manner (Gorman. 1991). Kernel infection by A. Jlavus and aflatoxin contamination in maize are greatly influenced by many environmental factors, such as insect incidence, temperature, humidity, water stress. Genotypes, inoculation techniques, inoculum (Aspergillus species), and kernel sampling procedure may also play important roles. Genetic studies on more diversified maize germplasms are urgently needed. Ear Moisture Lose Rate in Maize

Ear Development and Ear Moisture Loss Rate in Maize

Ear development has been divided into four naturally distinct stages (Shaw and Loomis. 1950): 1) ear shoot development, pollen formation, and fertilization;

2) rapid growth of ear shoot, during which the husk dimensions of the ear attain their maximum size; 3) physiologic maturity at 30 to 40% grain moisture content; and 4) drying of the physiological mature ears to about 20% moisture. Ear development during the first three stages affects yield directly, and the fourth stage is important in crop production in relation to possible frost damage at harvest time.

The rate of the ear drying process is important for both the farmer and the seed producer to determine whether field drying or artifical drying is used. A fast drying hybrid can help farmers avoid yield loss caused by delayed harvesting, lodging, dropped ears, bird damage, and diseases and reduce artificial grain drying cost. Early harvest and artificial drying of seed corn using forced heated air are necessary to avoid frost injury in northern areas of the USA and to avoid damage due to insects and field weathering (Bdliya and Burris. 1988). However, exposure to high temperature during artificial drying of high moisture seed corn may result in reduced seed quality and lead to poorer germination, reduced stand, and lower seedling vigor (Bdliya and Burris, 1988). Harrison and Wright (1929) found that seeds harvested at 39.6% moisture had reduced germination when they 39 were dried at 50 C and that drying such seeds at 60 or 70 C had prevented germination of the seeds.

Aldrich (1943) defined physiologic maturity as the point at which maximum dry weight of the grain is attained. Shaw and Thom (1951) indicated that the average length from silking to physiologic maturity in maize in Iowa was

51 days with a relatively small variation and no significant differences between early and late varieties. This suggests that the period of development from silking to physiologic maturity is relatively independent of weather variations. However, other studies (Andrew et al., 1956; Dessureaux et al., 1948) indicate a large variation caused by weather for this period. After physiological maturity, the rate of moisture loss is more dependent on weather than any other factor (Aldrich et al., 1975). Kang et al. (1986) found that hybrids with a relatively higher moisture reduction rate will also be fast fillers. Thus, they suggested that more emphasis should be given to moisture reduction during the filling period and that final harvest moisture may be used as a selection criterion to select maize with faster dry-down rale.

Weather Factors Influencing Ear Moisture Loss Rate in Maize

Ear drying in the Held is affected by temperature, humidity, and other weather factors (Schmidt and Hallauer, 1966; Aldrich et al.. 1975; Kang et al.,

1983, 1986). Gunn and Christensen (1965) noted high correlations between the number of effective degree days to midsilk and the moisture percentages of ears 40 at all stages of development. Hallauer and Russell (1961) conducted a three-year study to estimate the rate of grain-moisture reduction from 40 days after silking to approximate physiological maturity and to determine the effects of six weather factors, viz., pan evaporation data, wind movement, relative humidity, duration of sunshine, precipitation, and degree days. They found that none of the factors, except degree days, showed a consistent association with grain-moisture loss and that the moisture reduction was influenced by the weather variations among years. They concluded that the use of any one of the six weather factors for estimating grain-moisture reduction was unreliable for the period studied.

Schmidt and Hallauer (1966) showed that above 30% kernel moisture content, the rate of moisture reduction was significantly related to the temperature of the air and below 30% kernel moisture content, the rate of moisture reduction was significantly related to the humidity of the air.

Agronomic Trails Related to Ear Moisture Loss Rate in Maize _____

Certain agronomic traits seem to be related to ear moisture loss rate.

Significant phenotypic and/or genotypic correlations between ear moisture loss rate and agronomic traits of maize, such as husk tightness, husk weight and number, cob weight and length, kernel row number and depth, and pericarp permeability, have been reported (Purdy and Crane, 1967b; Troyer and

Ambrose, 1971; Kang et al., 1975; Hicks et al., 1976; Cavalieri and Smith,

1985). Kiesselbach (1950) indicated that husks protected grain viability against injury due to light freezing temperatures in the fall, but husks also retarded the

drying rate of the grain after maturity. Troyer and Ambrose (1971) reported that

husks limited air movement around the grain and that a low number of loose,

short husks was conducive to fast grain-drying. Hicks et al. (1976) found that

loose husks caused faster cob, grain, and ear drying compared to normal or tied

husks. Kang et al. (1975, 1983, 1986) found that a significant positive correlation

existed between husk weight and ear moisture content and that reduced husk

weight would be expected to reduce grain moisture at harvest. Cross et al. (1987)

divergently selected live synthetics for two or more cycles for relative rates of

moisture loss from ears in the laboratory. The selected substrains were evaluated

in six environments along with substrains divergently selected for two cycles for

ear moisture content at 45 d postpollination, They found that selection for slow

relative moisture loss in the laboratory changed Held harvest moisture by -6.73

g kg 1 cycle 1 and produced correlated reductions in ear length, kernels per ear,

kernel rows per ear, ear weight, and root lodging. The selection for low ear

moisture at 45 d postpollination changed harvest ear moisture in selected strains

by -7.15 g kg ' cycle1 and resulted in correlated increases in test weight and decreases in stalk lodging. Selection for fast relative moisture loss in the

laboratory or high moisture content at 45 d poslpollination generally produced

increased ear moisture at harvest in selected strains. The lower harvest moisture

resulted from the selected strains having a lower moisture content at physiological 42 maturity rather than a faster drying rate. Results in this study suggested that direct selection for ear moisture content at or near physiological maturity may be a more efficient selection procedure for reducing ear moisture at harvest. Cross and Kabir (1989) showed that, in a late maturating maize diallel set, significant and negative correlations between rates of moisture loss and grain yield and ear length were obtained. A significantly positive correlation between rates of moistuiv loss and ear moisture were found. In an early maturing maize diallel set. significantly negative correlations between rales of moisture loss and kernel depth, kernel rows per ear, and shelling ratio were reported. They concluded that selection for both low moisture at harvest and fast rates of moisture loss might be more effective than considering either character alone.

Physical structure of the pericarp has been associated with grain drying rate. Purdy and Crane (1967b) reported that the pericarp was semi-permeable and was an important factor responsible for differential rales of moisture loss from kernels; faster drying rates being associated with thinner pericarps and greater permeability. Nass and Crane (1970) studied the effects of endosperm mutants on drying rale in maize hybrids. They found opaque-2 showed a significantly higher drying rate at first five harvests from 35 to 77 days after pollination; sugary-2 had a significantly lower drying rate than normal at first five harvests; floury-1 and wx did not differ significantly in drying rale from normal at any of the harvests. They concluded that drying rate was regulated in part by 43 hydrophilic compounds, such as sugars, proteins, and carbohydrates, in the endosperm of the maize kernel.

Hunter et al. (1979) found that dent hybrids had a higher moisture level at physiological maturity than flint hybrids. There was no major inherent difference in field drying rate between the flint and dent endosperm types. Plant defoliation would speed drying rate of ears (Troyer and Ambrose, 1971;

Tollenaar and Daynard. 1978).

Kang et al. (1983) indicated that heavier cobs and fewer growing degree days to mid-silk would aid in reducing grain moisture content at harvest. Kang et al. (198b) also found that rate of grain fill had a direct positive effect on grain moisture reduction per growing degree day during the filling period, which suggested that hybrids with a relatively higher grain moisture reduction rate would also be fast fillers.

Kang el al. (1978) developed a reliable, non-destructive method of measuring ear moisture loss rate (EMLR) in the field. They evaluated an electronic probe method by comparing probe readings with percent ear moisture determined by the oven method. A significant correlation coefficient between probe readings and percent ear moisture was established. Furthermore, the coefficient of variation for probe moisture (10%) was less than that for percent ear moisture obtained by the oven method (15.8%), indicating that precision in determining percent ear moisture was greater with the probe method than with 44 the oven method. Practical uses of this electronic probe (DC-10 moisture meter) have been reported (Kang and Zuber, 1987, 1989; Kondapi et al., 1993).

Freppon et al. (1992) also reported the use of another portable electronic meter,

JCS-1, for estimating ear moisture of maize in the field. They found that the moisture meter produced estimates of moisture content that correlated well in two years (r=0.85 and 0.82) with estimates obtained by the oven method. The DC-

10 moisture meter has a calibrated scale capable of measuring relative ear moisture between 6 and 70 (g 0.1 kg '), whereas the JCS-1 electronic meter measures moisture up to 45 (g 0.1 kg ‘).

Genetics of Ear Moisture Loss Rate in Maize

There have been only a few genetic studies on EMLR in maize.

Researchers have observed significant differences among inbred lines and hybrids under laboratory and field conditions and indicated that ear moisture loss rate was a heritable trait (Crane et al., 1959; Hillson and Penny. 1965; Purdy and Crane,

1967a; Nass and Crane, 1970; Cross, 1985; Hallauer and Russell 1962). Hillson and Penny (1965) indicated that hybrids lost moisture at different rates and that parent inbred lines differed in their effects in hybrids on the rate of moisture loss.

Purdy and Crane (1967a) studied inheritance of drying rale and found that additive genetic effects were more important than non-additive effects. Cross and

Kabir (1989) conducted two diallel studies on ear drying rates and reported that both GCA and SCA effects were significant, with the additive genetic component 45 being more important than the non-additive genetic component. Kang and Zuber

(1989) evaluated the comparative efleets of"2y, ly. and 0y gene dosages on grain moisture, probe moisture, husk moisture, and growing degree days to midsilk.

They found that GCA and SCA mean squares for probe moisture and grain moisture in both yellow x yellow and white x white endosperm diallels were significant. When overall comparisons were made, probe moisture was significantly higher for the 2y class than for the ly and Oy classes and that grain moisture was significantly higher for the 2y than for Oy class. They suggested that the y allele might have had a pleiotropic-dosage effect on these two traits.

Kondapi et al. (1993) studied two populations to elucidate the genetic nature of

EMLR. They used North Carolina design II and computed heritability. The result indicated that narrow-sense heritabilities for EMLR in the two populations were high, indicating a recurrent selection procedure should be effective in concentrating genes for this trait.

To date, it is clear that EMLR is a quantitative genetic trait and is also influenced b> en\ ironmcntal conditions. The EMLRs are different between midsilk to physiological maturity and alter physiological maturity. Future research should focus on studying the whole picture of EMLR from inidsilk to harvest and the relationship between EMLR and yield and other important agronomic traits. 46

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Zummo. N.. and G.E. Scott. 1992. Interaction of Fusarium moniliforme and Aspergillus Jlavus on kernel infection and allatoxin contamination in maize ears. Plant Dis. 76:771-773. CHAPTER 1. DIALLEL ANALYSIS FOR PERCENT KERNEL INFECTION BY ASPERGILLUS FLAVUS IN MAIZE

Introduction

Kernel infection by A. Jlavus Link ex Fries and subsequent preharvest afiatoxin contamination of maize (Zea m ays L.) kernels are two major and chronic production problems not only in the southeastern United States but also in other parts of the world (Nichols, 1983; Zuber and Darrah, 1987; Kang et al.,

1988). Effects of afiatoxin consumption on animal health range from decreased growth rates, reduced weight gain and reproductive efficiency to mortality

(Brown et al., 1981; Richard et al.. 1983; Cheeke and shull, 1985; Pier, 1987).

Aflatoxins are also of concern for human health, as they have been linked to liver cancer (Bodine and Mertens, 1983; Pier, 1987). Although efforts have been made to develop methods to prevent maize infection by Aspergillus spp. and to find detoxification methods to lessen afiatoxin contamination in maize, a viable solution has not been found (Lillehoj and Wall, 1987).

A. Jlavus is a saprophyte and completes a growth cycle from saprophytic development on decaying plant parts to facultative necrotrophy in living tissues

(Lillehoj, 1987). The initial source of primary inoculum in maize fields has been thought to be conidia dispersed by high altitude wind currents from farm buildings and grain storage bins or/and produced by mycelia grown from sclerotia that overwinter in plant debris and litter in the soil (Jones, 1979).

62 63

Conidia are either dispersed by wind or carried by arthropod vectors to maize ears (Wicklow, 1983) Growth of conidia or colonization is dependent on the conditions of the silks, temperature, humidity, and other factors (Marsh and

Payne. 1981: Davis and Diener. 1983). A. Jlavus can colonize maize silks, glume tissue, and kernels, without any damage to husks (Jones et al., 1980;

Payne, 1983).

The method for determining percent kernel infection (PKI) has been developed and modi lied by several researchers (Tucker et al., 1986; Scott and

Zummo. 1988; Zummo and Scott. 1989: Scott el al.. 1991). Significant correlations between PKI and actual allatoxin were found by researchs (Payne et al., 1988; Scott et al.. 1991; Tucker et al,. 1986). Hybrids that show resistance to PKI have significantly lower concentrations of allatoxin in the grain at harvest

(Scott et al.. 1991).

Host-planl resistance is considered to be the most logical and useful method of control (Gorman and Kang. 1991). Several researchers have indicated that resistance to allatoxin contamination is partly under genetic control (Zuber et al.. 1978: Widstrom el al.. 1984: 1987; Darrah et al.. 1987; Kang et al..

1990; Gorman and Kang. 1992). In most genetic studies, additive genetic effects were shown to be more important in determining allatoxin resistance than non­ additive effects (Zuber et al., 1978; Widstrom et al.. 1984: Darrah et al., 1987;

Gorman, 1991). However. Gardner et al. (1987) indicated that the reverse could 64

be true and could be attributed to different inoculation methods. Campbell and

White (1995) also found, by generation mean analysis, that in maize additive and

dominance gene actions controlled resistance to Aspergillus ear rot caused by A.

Jlavus. Although some progress in understanding the mechanisms of resistance

to afiatoxin concentration in maize kernel has been achieved, the genetics of

kernel infection by A. Jlavus remains poorly understood. Maize kernel infection

by A. Jlavus and subsequent allatoxin contamination are two consecutive

processes. A sound knowledge of genetics of kernel infection per se is needed to

solve the aflatoxion contamination problem in maize. Logicalcally, if kernel

infection by A. Jlavus can be reduced/prevented, allatoxin contamination can be

reduced/prevented. Additionally, resistance of maize to allatoxin contamination

has been linked to factors associated with ouler integuments of developing kernels

and other ear components inside the kernel (Darrah et al., 1987; Gao et al.,

1995). These factors are directly related to resistance of maize to kernel infection by zl. Jlavus, whereas the resistance of outer integument in maize is only

indirectl) related to allatoxin production via its resistance to kernel infection.

Recently. Gao et al. (1995) suggested that wax and cutin layers of maize kernel pericarps may play a role in resistance to afiatoxin contamination and that

resistance in some genotypes may be due to other preformed compounds inside the kernel as well. Quantification of PKI is a reliable method for predicting

relative allatoxin eonlration (Tucker et al., 1986). Thus, a greater understanding 65

of inheritance of PKI is very important and needed to facilitate development of

inbreds and hybrids with reasonably high levels of resistance to Aspergillus spp.

The main objective of this research was to determine GCA, SC A, and reciprocal

effects for PKI in a 10-parent diallel cross.

Materials and Methods

Ninety F, single crosses made from 10 inbred lines (L108, L266, L329,

L605. L654. L668. L729, Hill, Mo 17. and B73) were machine-planted into a

Commerce silt loam soil (fine-silty, mixed, nonacid, thermic Aerie Fluvaquent)

in single-row plots of 6.1 m length, with 102 cm row spacing and 30 cm plant

spacing within rows, on April 9. 1992 and on April 28. 1994. Experiments were

replicated four times at the Ben Hur Plant Science Farm, Baton Rouge, LA, in

a randomized complete block design.

Midsilk (50 % plants in a plot with silks emerged) dates were recorded for each plot. Ten days after midsilk. 10 ears per plot in 1992 and 5 ears per plot in

1994 were randomly selected and inoculated with 1 niL A. Jlavus conidial

suspension of isolate NRRL 3357 (10 x 10" conidia mL '), using a needle-in-silk channel technique (Scott and Zummo. 1988). Ten days later, the same ears were

re-inoculated with 1 mL A. jlavus conidial suspension (10 x 10" conidia m L 1).

Inoculated ears were harvested in early September in both years and shelled in

mid- December in 1992 and in mid-September in 1994 on a plot basis. 66

Kernels assayed for fungal infection were randomly selected from whole

kernels of all inoculated ears of each plot. The kernels were surface sterilized

by dipping them for 1 min in 70 mL/L aqueous ethanol, soaked for 3 min in

1.25% NaOCI (laundry bleach), and rinsed in sterile, distilled water (Scott et al.,

1991). Three hundred and ninety kernels per plot in 1992 and 156 kernels per

plot in 1994 were plated in 100-mm petri dishes (13 kernels per dish) on Czapek

solution agar amended with 7.5% sodium chloride to restrict growth of other

fungi and bacteria (Scott el al., 1991). After 6 d of incubation at 28 ± 2 C,

plates were examined for A. Jlavus growth and percent kernels infected with A.

Jlavus was computed.

Data were analyzed according to a general linear model (GLM), treating

both hybrids and years as fixed effects. Here, we treated years as a fixed factor

because in the two experimental years, many factors that influenced kernel

infection by A. Jlavus were not randomly selected. Firstly, planting date in 1992

was april 9 and in 1994 it had to be postponed to April 28 because of wet fields.

Secondly, in 1992. hurricane Andrew caused all hybrids to lodge and remain

under water for several days. Thirdly, experimental plots were shelled at

different intervals after harvesting in the two years. Non of these was a random

decision on our part. Therefore, the year effect was not random but was

confounded with planting date, hurricane damage, and shelling time. Least square means (Table 1.1) for each hybrid were used to compute

combining t.‘4 via Grifling Method .3 and Model I (1956). The ratio, 2Vl,

/(2VJ. + VJ. was calculated according to Baker (1978).

Results and Discussion

Analysis of variance for PKI revealed that GCA, SCA, and reciprocal

mean squares were significantly different (Table 1.2). The GCA, SCA, and

reciprocal effects should be used in designing a maize breeding program aimed

at enhancing maize resistance to A. jla vu s. The 2V,, /(2V,, + V J ratio was 0.54,

indicating that both GCA and SCA were important. Gorman and Kang (1991)

critically reviewed previous genetic research on the resistance of maize to

aflatoxin accumulation and reported that 2VU /(2Vtt + V j ratios are between

0.36 and 1.00. In our experiment, the inbred lines, inoculation technique, and

environments differed from those of previous studies. A more important

difference between this study and previous investigations is that resistance to A.

Jlavus infection was evaluated, and not resistance to aflatoxin accumulation.

The GCA x year. SCA x year, and reciprocal x year interactions were

significant. These results not only indicated that different planting dates and

storage of ears alter harvest for different time periods might influence resistance

of maize genotypes to kernel infection by A. jla vu s. but also strongly suggested

that multi-year experiments should be conducted to obtain more reliable genetic

information. 68

The GCA effects of inbred lines L108, L605, L654, and Hill were significantly negative, whereas those of inbred lines L329. L729, and B73 were significantly positive (Table 1.1). When GCA effects were compared with common parent means, parents with significant negative GCA effects had lower

PK1, whereas parents with significant positive GCA effects generally showed a relatively high PKI.

The F, crosses LI08 x Mol7. L329 x HI 11, L654 x L266, and Mol7 x

B73 showed significant negative SCA effects (Table 1.3). The corresponding

PKIs for these crosses were low. The F, crosses L266 x L729, L329 x Mol7,

L605 x Mo 17. L654 x L329, L668 x L729. and L729 x Mo 17 had significant, positive reciprocal effects, suggesting that their reciprocals should be beneficial in a breeding program. The F, crosses L654 x B73 and L668 x L329 showed significant, negative reciprocal effects. The common parent means for PKI (Table

1.3) showed that a relatively low PKI was always associated with either a significant, negative SCA effect or a significant, negative reciprocal effect; which suggested that the SCA and reciprocal effects were important in lowering PKI in maize. It also was interesting that PKI was always greater when Mol7 was a male parent in a cross than that when Mo 17 was a female parent in a cross

(Table 1.1). The PKI mean of common male Mol7 was 21.8, whereas that of common female Mol7 was 13.4, i.e., 38.5% lower than the former. Inbred line

L668 also showed a similar result, i.e., PKI mean of common female L668 69

(16.4) was 23.7% lower than that of common male L668 (21.5). Therefore, the use of these two inbred lines as female parents, in general, should help in lowering PKI. In general, the use of inbred lines L226. L729, and B73 as female parents should be avoided.

The coefficient of variance (CV) for this experiment was 52%, which was relatively high. However, this value falls in the range reported in other A. Jlavus infection or aflatoxin contamination research (Widstrom et al.. 1984; Scott et al.,

1991). The underlying reason for such high CVs may be that A. Jlavus infection, colonization, and aflatoxin accumulation in maize need specific environmental conditions (Jones el al., 1980; Payne et al., 1983; Lillehoj, 1987). But when maize ears are inoculated, suitable environmental conditions may not appear for hours or even days. The real situation is that most environmental conditions in the field are beyond our control. Another possible reason is that resistance of maize kernels to infection by A. jla vu s is composed of the outer kernel integument and chemical(s) inside the kernels. The kernels evaluated in this experiment were the second generation, open-pollinated seed harvested from F, plants. This approach is similar to that used by other experiments (Gardner, et al., 1987; Gorman et al.,

1992;Widstrom el al.. 1984; Zuber et al., 1978). Embryos(2n) and endosperms

(3n) of such seeds have different genotypes because of different males (open pollination) and gene segregation. These difference genotypes could be expected to behave differently relative to resistance to kernel infection by A. Jlavus. I

Table 1.1. Mean percent kernel infection by Aspergillus Jlavus for 90 F, crosses, and general combining ability (GCA) effects and common parent means for 10 inbred lines. F, crosses were evaluated at Baton Rouge, LA in 1992 and 1994.

Parent lines L654 L668 L108 L266 L729 L329 L605 Hill Mol7 B73 Common parent mean GCA effects

L654 16.5 10.3 7.2 12.4 30.4 12.2 7.5 13.3 16.9 13.9 -4.07"

L668 14.8 14.9 14.7 12.6 13.5 18.0 16.1 22.0 21.1 19.0 1.69

LI 08 10.7 17.7 16.1 21.8 23.5 8.6 12.1 6.9 15.7 14.4 -3.44"

L266 10.3 23.9 17.7 26.1 17.2 16.7 14.8 21.5 26.6 18.2 0.79

L729 16.2 34.7 19.7 14.4 22.5 21.5 20.0 35.1 24.0 21.2 4.26"

L329 14.5 35.0 14.2 19.2 25.4 14.9 11.1 26.2 18.4 19.3 2.10*

L605 10.0 18.3 16.1 10.2 18.3 12.4 7.2 25.4 16.3 15.0 -2.76"

(Table con’d)

-o o I

Hill 6.5 17.6 10.7 18.9 23.7 12.7 14.4 26.2 16.6 15.7 -2.03’

Mo 17 8.0 12.2 6.4 16.3 19.0 15.7 8.1 23.2 12.0 17.6 0.22

B73 31.2 17.3 15.8 34.8 14.8 20.7 21.6 22.2 20.0 20.3 3.24”

*. ** Significant at 0.05 and 0.01 probability levels, respectively. LSD0()

Table 1.2. Analysis of variance for percent kernel infection by Aspergillus flavus for 90 F, crosses of maize evaluated at Baton Rouge, LA in 1992 and 1994.

Source DF Mean square

Years (Y) 1 47754.397“

Reps within Y 4 141.301

F, crosses (C) 89 264.532“

GCA 9 808.616"

SCA* 35 208.757“

Reciprocal (Ree.) 45 202.749“

C x Y 89 237.114“

GCA x Y 9 653.523“

SCA x Y 35 237.510“

Rec. x Y 45 153.524“

Error 353 81.535

Coefficient of variation: 51 .9%; R-square: 76.5%.

*, ** Significant at 0.05 and 0.01 probability levels, respectively. + General combining ability. * Specific combining ability. 73

Table 1.3. F, crosses with significant specific combining ability (SCA) and/or significant reciprocal effects. F ( crosses were evaluated at Baton Rouge, LA in 1992 and 1994.

F, crosses SCA effect Reciprocal effect F, cross mean

L108xM ol7 -7.54** 0.25 6.9

L266xB73 9.24** -4.13 26.6

L266xL729 -2.23 5.86* 26.1

L329xH 111 -5.59* -0.78 11.1

L329xM ol7 1.13 5.34* 26.2

L605xM ol7 1.80 8.64** 25.4

L654xL266 -5.42* -1.53 7.2

L654xL329 6.95** 7.96” 30.4

L654xB73 7.41” -7.16” 16.9

L668xL329 3.05 -10.75” 13.5

L668xL729 0.23 5.86* 26.1

L729xM ol7 5.15* 8.08” 35.1

L729xB73 9.24** 4.59 24.0

HI 1 lxMol7 9.07” 1.45 26.2

Mol7xB73 -4.88* -4.21 12.0

*,** Significant at 0.05 and 0.01 probability levels, respectively. LSD00, and LSD,)0I for testing SCA effects being different from zero were 4,51 and 5.92, 74 respectively. LSD00, and LSD001 for testing differences between s^ and sik were 6.76 and 8.88, respectively. LSD0()5 and LSD001 for testing differences between Sy and skl were 6.26 and 8.22, respectively. LSD0.(* and LSD00i for testing reciprocal effects were 5.11 and 6.72, respectively. 75

References

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Brown, R.D.. A.C. Pier, J.L. Richard., and R.E. Krogstad. 1981, Effects of dietary aflatoxin on existing bacterial intrammary infections of dairy cows. Am. .1. Veterinary Res.42:927-939.

Campbell, K.W., and D.G. White. 1995. Inheritance of resistance to Aspergillus ear rot and aflatoxin in corn genotypes. Phytopathology 85:886-896.

Cheeke, P.R., and L.R. Shull. 1985. Natural toxicants in feeds and poisonous plants. AVI Publishing Company, Westport, CT.

Darrah, L.L., E.B. Lillehoj, M.S. Zuber, G.E. Scott, D. Thompson, West, D. R.. N.W.Widstrom, and B.A. Forimtm. 1987, Inheritance of aflatoxin B, levels in maize kernels under modified natural inoculation with Aspergillus Jlavus. Crop Sci. 27:869-872.

Davis, N.D., and U.L. Diener. 1983. Some Characteristics of toxigenic and nontoxigenic isolates of Aspergillus Jlavus and Aspergillus parasiticus, p.l- 5. In U.L. Diener, R.L. Asquith, and J.W. Dickens, (ed.) Aflatoxin and Aspergillus Jlavus in corn. So. Coop. Ser. Bull. 279. Auburn Univ., AL.

Gao, B.Z.. J. S. Russin, T.E. Cleveland, R.L.Brown, and N.W. Widstrom 1995. Wax and cutin layers in maize kernels associated with resistance to aflatoxin production by Aspergillus Jlavus. J. Food Production 58:296-300.

Gardner, C.A.C.. L.L. Darrah. M.S. Zuber, and J.R. Wallin. 1987. Genetic control of aflatoxin production in maize. Plant Dis. 71:426-429.

Gorman, D.P. 1991. Genetics of resistance to preharvest aflatoxin contamination in maize containing the Lfy gene. Diss. Abstr. AAC 9200062.

Gorman, D.P., and M.S. Kang. 1991. Preharvest aflatoxin contamination in maize: Resistance and genetics. Plant Breeding. 107:1-10. 76

Gorman, D. P., M.S. Kang, T. Cleveland, and R.L. Hutchinson. 1992. Field aflatoxin production by Aspergillus jlavus and A. parasiticus on maize kernels. Euphytica 61:187-191.

GrilTing. B. 1956. Concept of general and specific combining ability in relation to dial lei crossing systems. Aust. J. Biol. Sci. 9:463-493.

Jones, R.K. 1979. The epidemiology and management of aflatoxins and other mycotoxins. p.381-392. In J.G. Horsfall, and E.B. Cowling, (ed.). Plant Disease, vol. IV. Academic Press, New York.

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Kang, M.S., E.B. Lillehoj. J.G. Marshall, and W. Hall. 1988. Preharvest aflatoxin levels in corn hybrid kernels in Louisiana. Cereal Res. Coinmun. 16:237-244.

Kang, M.S., E.B. Lillehoj, and N.W. Widstrom. 1990. Field aflatoxin contamination of maize genotypes of broad genetic base. Euphytica 51:19- 23.

Lillehoj. E.B. 1987. The atlatoxin-in-maize problem: the historical perspective, p. 13-32. In M.S. Zuber, E.B. Lillehoj, and B.L. Renfro, (ed.). Aflatoxin in maize. A proceedings of the workshop. CIMMYT, Mexico, D.F.

Lillehoj, E.B., and J.H. Wall. 1987. Decontamination of aflatoxin-contaminated maize grain, p.260-279. In M.S. Zuber, E.B. Lillehoj, and B.L. Renfro, (ed.). Aflatoxin in maize. A proceedings of the workshop. CIMMYT, Mexico, D.F.

Marsh. S. F.. and G. A. Payne. 1981. Scanning electron microscopy of Aspergillus jlavus infection of corn silks. Phytopathology 71:893. Abstr.

Nichols, T.E. 1983. Economic effects of aflatoxin in corn. p. 67-71. In U.L. Diener, R.L. Asquith, and J.W. Dickens, (ed.), Aflatoxin and Aspergillus jla vu s in corn. So. Coop. Ser. Bull. 279. Auburn Univ., Auburn, AL.

Payne,G.A. 1983. Nature of Field Infection of Corn by Aspergillus jlavus. p. lb- 19. In U.L. Diener. R.L. Asquith, and J.W. Dickens, (ed.), Aflatoxin 77

and Aspergillus Jlavus in corn. So. Coop. Ser. Bull. 279. Auburn Univ., Auburn. AL.

Pier, A.C. 1987. Aflatoxicosis and immunosuppression in mammalian animals, p.58-65. In M.S. Zuber, E.B. Lillehoj, and B.L. Renfro, (ed.). Aflatoxin in maize. A proceedings of the workshop. CIMMYT, Mexico, D.F.

Richard, J.L., A.C. Pier, R.D. Stubblefield, R.L. Lyon., and R.C. Cutlip. 1983. Effect of feeding corn naturally contaminated with aflatoxin on feed efficiency, physiologic, immunologic and pathologic changes and on tissue residue in steers. Am. J. Veterinary Res. 49:1294-1299.

Scott. G .E.. and N. Zummo. 1988. Sources of resistance in maize to kernel infection by Aspergillus Jlavus in the field: Crop Sci. 28:504-507.

Scott, G.E., N. Zummo, E.B. Lillehoj, N.W. Widstrom, M.S. Kang, D.R. West, G.A. Payne, T.E. Cleveland, O.H. Calvert, and B.A. Fortnum. 1991. Aflatoxin in corn hybrids field inoculated with Aspergillus flavus. Agron. J. 83:595-598.

Tucker. D.H.. Jr.. L.E. Trevathan, S.B. King, and G.E. Scott. 1986. Effect of four inoculation techniques on infection and aflatoxin concentration of resistant and susceptible corn hybrids inoculated with Aspergillus Jlavus. Phytopathology. 76:290-293.

Wicklow, D.T. 1983. Taxonomic features and ecological significance of sclerotia. p.6-12. hi U.L. Diener, R.L. Asquith, and J.W. Dickens, (ed.), Aflatoxin and Aspergillus Jlavus in corn. So. Coop. Ser. Bull. 279. Auburn Univ., Auburn, AL.

Widstrom. N.W., W.W. McMillian, and D.M. Wilson. 1987. Segregation for resistance to aflatoxin contamination among seeds on an ear of hybrid maize. Crop Sci. 27:961-963.

Widstrom, N.W., D.M. Wilson, and W.W. McMillian. 1984. Ear resistance of maize inbreds to field aflatoxin contamination. Crop Sci. 24:1155-1157.

Zuber. M.S.. O.H. Calvert. W.F. Kwolek, E.B. Lillehoj, and M.S. Kang. 1978. Aflatoxin B, production in an eight-line diallel of Zea mays infected with Aspergillus jlavus. Phytopathology 68:1345-1349. 78

Zuber, M.S., and L.L. Darrah. 1987. International survey on natural aflatoxin occurrence in maize, p.285-288. In M.S. Zuber, E.B. Lillehoj,and B.L.Renfro, (ed.). Aflatoxin in maize. A proceedings of the workshop. CIMMYT, Mexico, D.F.

Zummo. N.. and G.E. Scott. 1989. Evaluation of Field inoculation techniques for screening maize genotypes against kernel infection by Aspergillus Jlavus in Mississippi. Plant Dis. 73:313-316. CHAFFER 2. A DIALLEL ANALYSIS OF EAR MOISTURE LOSS RATE IN MAIZE

Introduction

Enhancing ear moisture loss rate (EMLR) in maize is an important

breeding objective to reduce growers’ production costs relative to artificial grain

drying and economic losses associated with delayed harvesting, such as lodging,

dropped ears, bird damage, and diseases due to leaving maize in the field longer

for natural dry-down (Purdy and Crane. 1967a; Nass and Crane, 1970; Kang et

al.. 1978). Maize hybrids with rapid ear moisture loss rate (EMLR) are needed

especially in maize production areas with a humid climate during the grain

maturation period and the grain dry-down period following physiological maturity.

Ear drying in the field is affected by temperature, humidity, and other

weather factors (Schmidt and Hallauer, 1966; Aldrich et al., 1975; Kang et al.,

1983. 1986). Significant phenotypic and/or genotypic correlations between EMLR

and agronomic traits of maize, such as husk tightness, husk weight and number,

cob weight and length, kernel row number and depth, and pericarp permeability,

have been reported (Purdy and Crane. 1967b; Troyer and Ambrose, 1971; Kang et al., 1975; - Hicks et al.. 1976; Cavalieri and Smith, 1985).

Kang et al. (1978) developed a reliable, non-destructive method of

measuring EMLR in the field. Practical uses of this electronic probe (DC-10

moisture meter) have been reported (Kang and Zuber. 1987. 1989; Kondapi et al..

79 80

1993). Freppon et al. (1992) also reported the use of another portable electronic meter, JCS-1, lor estimating ear moisture of maize in the field. These studies showed that correlation between ear moisture content (oven method) and probe moisture readings was high (r= 0 .8 2 to 0.85) and indicated that both meters were reliable. The D C-10 moisture meter has a calibrated scale capable of measuring relative ear moisture between 6 and 70 (g 0.1 kg '), whereas JCS-1 measures moisture up to 45 (g 0 .1 kg ').

There have been only a few genetic studies on EMLR in maize.

Researchers have observed significant differences among inbred lines and hybrids under laboratory conditions and indicated that EMLR was a heritable trait (Crane et al., 1959; Hillson and Penny. 1965; Purdy and Crane. 1967a; Nass and Crane,

1970; Cross. 1985). Purdy and Crane (1967a) and Cross and Kabir (1989) conducted diallel studies on ear drying rates and reported that both GCA and SCA effects were significant, with the additive genetic component being more important than the non-additive genetic component. Cross (1985) developed a mass selection method based on estimation of ear moisture loss in the laboratory. Cross and his co-researchers (1987. 1989) concluded that selection of inbred lines based on low ear moisture al 45 d post-pollination could effectively reduce ear moisture at harvest. Ear moisture differences at physiological maturity were deemed important

(Cross. 1985). Kondapi el al. (1993) reported narrow-sense heritabilities for

EMLR in two populations to be high, indicating a recurrent selection procedure 81 should he effective in concentrating genes for this trait. Additional genetic

information is needed on field EMLR, especially for germplasms for potential use in the southern United States,

The objectives of this investigation were to determine GCA, SCA, and reciprocal effects for EMLR estimated in the field with the electronic probe device of Kang et al. (1978) in a 10-parent dial lei mating design and to identify inbred lines that could be used in a breeding program to lower harvest moisture of maize. In addition, we wanted to detect any relationship between EMLR and

PKI.

Materials and Methods

The same 10 inbred lines as used by Kang el al. (I995)were used in this investigation. Inbred lines were crossed to produce a complete diallel set (90 F, hybrids, including reciprocals). The L lines (all full-season maturity) were developed al Louisiana State University and inbred lines Hill. Mol7, and B73

(early to medium maturity) were included as out-of-state reference lines. Seeds of the 90 F, hybrids were machine-planted into a Commerce silt loam soil (fme- silty, mixed, nonacid, thermic Aerie Fluvaquent) in single-row plots of 6.1 m length, with 102 cm inter-row spacing and 30 cm intra-row spacing, on 9 April

1992 and on 2N April 199-1. Experiments were replicated four times at the Ben

Hur Plant Science Farm. Baton Rouge. LA, in a randomized complete-block design. 82

Mitlsilk (50% plants in a plot with silks) dates were recorded for each plot.

First probe moisture (30DPM) was recorded 30 d after midsilk, on 8 randomly selected plants in 1992 and 5 randomly selected plants in 1994. Second probe moisture (51DPM) was recorded 51 d after midsilk on the same ears as used for

30DPM. EMLR was computed as follows: EMLR = (3()DPM-51 DPM)/21.

Plot means for 30DPM, 5IDPM , and EMLR were subjected to statistical analyses. To remove variances caused by different maturities of F, hybrids and by the differential weather conditions that prevailed during the different 21-d periods between 30DPM and 51DPM for different plots, many covariance models were tried and the one considered most suitable was a linear model with midsilk date and GDDs between 30DPM and 51DPM as covariatcs. Thus, the applicable general linear model for the data set was:

Y h f ‘h„ f * v„ + m ' +(J I *,,,,11.1 + 2 XiimloT e ||1Mk

V„ =g. + & +S.( + r„ • where V k = observed EMLR (g 0.1 kg'1 d *), /a = overall mean, a m= year effect. bkm = replication effect in each year. v„ = F, hybrid effect, avijm = interaction effect of F, hybrids with years, /i, - regression coefficient for the relation between Y and Xl|ll1kl , jj2 = regression coefficient for the relation between Y and X1|mk: . XMmkl = no. of days from planting to midsilk, X(|lllk2 =

GDDs between 30DPM and 51DPM. G,imk = residual effect, g, = GCA effect of inbred parent i. g, = GCA effect of inbred parent j. and s„ = SCA effect for /yth 83 cross, and rj( = reciprocal effect for ijih or j i th cross. For the ANCOVA of

30DPM and 5IDPM , the same model was used as for EMLR.

Adjusted means for 30DPM, 51DPM, and EMLR for F, hybrids were calculated (Table 2.1). Griffing’s diallel Method 3 (Model 1, i.e, genotypes considered fixed effects) was used to compute GCA, SCA, and reciprocal effects based on the adjusted means. Since data were analyzed according to a fixed model, estimation of components of variance and narrow-sense heritability was not appropriate (Griffing. 1956: Hallauer and Miranda, 1988). However, according to Baker (1978), components of mean squares, for a fixed effect model, can be used to compute the 2VM / (2V^, + Vs) ratio. This ratio is useful in dctcrmincing relative importance of GCA and SCA effects.

Statistical significance of F, hybrids was tested against the F,-hybrid x year interaction. The GCA. SCA. and reciprocal mean squares were tested against

GCA x year, SCA x year, and reciprocal x year interactions, respectively (Table

2.2). Adjusted plot means were used for comparing differences between L lines and out of stale lines for 30DPM. 5IDPM . and EMLR. Variance components were used to compute phenotypic and genotypic correlations. All statistical analyses were conducted using SAS (SAS Institute, Cary, NC).

Results and Discussion

The effectiveness of the covariance model used was determined by comparing the error variances of the covariance model and the regular model 84

(without covariates) (Neter cl al,, 1990). The covariance model reduced the error variance of the regular model by 7.5%.

The ANCOVA for EMLR revealed that the Fr hybrid and GCA mean squares were highly significant, whereas the SCA mean square was not significant

(Table 2.2). The 2VS / (2Vj, + VJ ratio was 0.81, which also indicated that GCA was more important than SCA. This result is consistent with previous research

(Purdy and Crane, 1976a; Cross and Kabir, 1989). Obviously, if this ratio =

1, the progeny performance could be predicated on the basis of GCA alone.

The F, hybrid. GCA. anil SCA mean squares lor 30DPM and 51DPM were all significant (Table 2.2). These results indicated that 30DPM and 5IDPM also are genetically controlled traits. The GCA effects for EMLR of inbred lines

L108, L605, and L654 were significantly positive, whereas, that of inbred line

Mol7 was significantly negative (Table 2.3). Hill and B73 had significant, negative GCA effects for 30DPM. L654. L668. Hill, and B73 showed significant, negative GCA effects for 5IDPM. Both GCA and SCA effects varied across years for 30DPM. 51DPM. and EMLR (Table 2.2), which suggested that data from additional years or environments would make GCA and SCA effects more reliable.

'fhe reciprocal mean square was highly significant for EMLR (Table 2.2).

F, hybrids LI 08 x Mo 17 (0.10* ). L329 x Mo 17 (0.19** ). and L605 x Mo 17

(0.1 1*), had significant, positive reciprocal effects, whereas F, hybrids L108 x 85

Hill (-0.13" ), L668 x L605 (-0.1 F), and L668 x L729 (-0.17" ), showed significant, negative reciprocal effects. The reciprocal x year mean squares for

51DPM and EMLR were not significant, indicating that they were more consistent

than GCA and SCA effects across years (Table 2.2).

The EMLR in this experiment was measured between 30 to 51 d after

midsilk. Cross et al. (1987) found that selection for low ear moisture at 45 d

postpollination effectively changed harvest ear moisture, in selected strains, by

-7.15 g kg 1 cycle1. We found a highly significant genetic correlation (0.87") between 51DPM and grain moisture at harvest (unpublished data from our lab).

Th us, 51DPM could be used as a criterion for indirect selection for low grain moisture at harvest. Selection for relatively high EMLR and low 30DPM should also be employed simultaneously to reduce 5IDPM and, in turn, harvest grain moisture as is evident from the following discussion. For example, F[ hybrid

L668 x L605 meets three selection criteria, i.e., EMLR is relatively high, both

L668 and L605 have significantly positive GCA effects for EMLR, 30DPM is relatively low. and thus. 51DPM has a relatively low value (31.1 g 0,1 kg '). In contrast. F, hybrid L668 x L I08 has a relatively high 5IDPM . even though its

EMLR is relatively high and both inbred lines have significantly positive GCA effects for EMLR. The high 5IDPM . therefore, could be attributed to a relatively high 30DPM. From the standpoint of EMLR, Mol7 x B73 was the most undesirable F, hybrid, as one of its parents. Mol7. showed significantly 86

negative GCA effects for EMLR, but its 5IDPM was relatively low because of

its low 30DPM.

F, hybrids LI08 x B73, L605 x L668, L654 x HI 11, L6S4 x L668, L668

x Hill, and MoI7 x L654 had relatively low 51DPM (Table 2.1), which is because the inbred lines involved in these hybrids either have significantly

negative GCA effects for 30DPM and 51DPM or have significantly positive GCA effects for EMLR or both. Thus, these hybrids should be useful in a breeding program for developing maize with low harvest ear moisture.

A comparson of the L inbred lines with the three out-of-state inbred lines for 30DPM. 51DPM, and EMLR (Table 2.4). indicating that the L inbred lines had significantly higher 30DPM. 51DPM, and EMLR than did the out-of-state lines. Although Lb68 had significantly higher 30DPM than inbred lines Hill and

B73, the mean 51DPM of L688 was not different from those of Hill and B73

(Table 2.1). Thus, L688 as well as Hill and B73 could be good germplasm sources for a breeding program aimed at lowering 51DPM and. in turn, lowering the harvest moisture of maize.

No significant phenotypic correlations among 30DPM, 5IDPM, EMLR and percent kerne! infection (PKI) were not observed. No significant genotypic correlations between 30DPM and PKI and between EMLR and PKI were found.

However, a significant genetic correlation between 5IDPM and PKI (r=0.67**) was detected. This result may suggest that the ear developmental stage at which 87

51DPM was recorded (around black layer) is a critical stage for A. jlavus

infection in maize kernels. This finding is consistent with a previous study that

reported that internal kernel infection did not occur until kernel moisture was

below 32% (Payne, 1987). This significant genetic correlation suggested that low

51DPM and low PKI could be achieved simultaneously.

We concluded that EMLR, 30DPM, and 51DPM were genetically

controlled traits. To select for low 51DPM, high EMLR, positive GCA effects for

EMLR, and a relatively low 30DPM were important. The GCA effect was more

important than SCA effect for determining EMLR. Extranuclear component(s)

contributed to enhancement of EMLR. Inbred lines L668. HI 11, and B73 could

be used as genetic sources in a maize breeding program for lowering 51DPM and grain moisture at harvest. Another benefit of selecting for a high EMLR could

be indirect selection for a high rate of grain-fill, as Kang et al. (1986) found a

positive correlation between these two attributes. Low 51DPM and low PKI could be achieved simutaneously. !

Table 2.1. Adjusted F, hybrid means and line means for first probe moisture (30DPM) and second probe moisture (51DPM) (g 0.1kg1) and ear moisture loss rate (EMLR) (s 0.1kg 1 d'1) for a 10-parent maize diallel cross grown at Baton Rouge. LA in 1992 and 1994.

Parentt ^\c?) L654 L668 L108 L266 L729 L329 L605 Hill M ol7 B73

L654 30DPM 52.8 64.8 54.2 61.6 62.1 60.2 51.6 56.7 56.3 51DPM 30.0 40.2 32.8 37.0 38.3 36.4 30.8 31.1 34.1 EMLR 1.08 1.17 1.02 1.17 1.13 1.13 0.99 1.22 1.06 L668 30DPM 50.9 66.1 53.6 53.7 60.0 55.3 53.3 49.7 49.3 51 DPM 30.3 38.4 31.6 35.4 36.6 33.0 29.2 34.2 28.7 EMLR 0.98 1.32 1.05 0.87 1.11 1.07 1.14 0.74 0.98 LI 08 30DPM 67.3 67.6 57.8 61.0 70.0 67.1 58.4 60.9 58.5 51 DPM 43.7 38.8 35.1 40.1 48.0 41.8 38.3 37.0 34.2 EMLR 1.12 1.37 1.08 1.00 1.05 1.20 0.96 1.14 1.16 L266 30DPM 53.1 51.7 57.2 60.0 60.4 56.6 52.1 50.6 49.6 51 DPM 32.4 32.3 36.8 38.1 40.7 37.0 31.4 35.9 30.7 EMLR 0.98 0.92 0.97 1.04 0.94 0.94 0.99 0.70 0.90 L729 30DPM 63.2 60.4 63.3 61.7 65.1 62.0 52.3 53.0 55.1 51 DPM 37.3 34.8 42.5 38.1 42.5 41.5 33.4 38.6 33.1 EMLR 1.23 1.22 0.99 1.12 1.08 0.97 0.90 0.69 1.05

(Table con’d)

00 oo I

L329 30DPM 63.0 57.6 69.1 67.1 64.3 66.8 52.5 59.5 55.6 51 DPM 38.6 36.8 44.7 49.7 40.8 41.8 33.7 40.2 35.5 EMLR 1.16 0.99 1.16 0.83 1.12 1.19 0.90 0.92 0.96 L605 30DPM 60.9 58.1 63.8 58.7 61.9 63.3 54.1 54.1 54.9 51 DPM 38.2 31.1 40.1 35.8 38.5 39.7 32.6 32.6 33.9 EMLR 1.08 1.28 1.12 1.09 1.11 1.12 1.03 1.03 1.00 Hill 30DPM 50.7 51.7 61.1 52.0 53.7 56.9 57.7 48.1 47.7 51 DPM 29.4 30.7 35.3 31.3 33.8 36.4 35.4 33.0 28.0 EMLR 1.01 1.00 1.23 0.99 0.95 0.98 1.06 0.72 0.94 Mo 17 30DPM 52.5 50.8 54.2 51.4 52.6 54.5 50.2 46.9 44.7 51 DPM 28.4 36.1 34.5 33.1 37.2 43.3 33.1 33.5 31.7 EMLR 1.15 0.70 0.94 0.87 0.73 0.53 0.81 0.64 0.62 B73 30DPM 55.1 51.7 57.4 50.1 53.6 54.7 52.9 50.0 43.8 51 DPM 31.0 28.5 31.7 29.4 32.1 37.4 32.8 29.7 34.4 EMLR 1.15 1.10 1.22 0.98 1.03 0.82 0.96 0.97 0.44 Line mean 30DPM 57.6 55.3 62.6 55.5 58.8 61.3 58.8 52.8 51.9 52.2 51 DPM 34.4 31.6 39.0 35.1 37.5 40.2 36.4 32.6 34.9 32.0 EMLR 1.10 1.05 1.12 0.96 1.02 1.00 1.07 0.97 0.81 0.96

Tukey critical values for testing differences in 30DPM among line means and 90 F( hybrids with 90% confidence =1.51 and 5.46, respectively. Tukey critical values for testing differences in 51 DPM among line means and 90 F, hybrids with 90% confidence =1.65 and 5.97, respectively.

oo o Tukey critical values for testing differences in EMLR among line means and 90 F, hybrids with 90% confidence =0.088 and 0.319. respectively.

vO o 91

Table 2.2. Analyses of variance for ear moisture loss rate (EMLR), first probe moisture (30DPM), and second probe moisture (51 DPM) in maize grown at Baton Rouge, LA in 1992 and 1994.

Mean squares

Source df EMLR 30DPM 51 DPM

(g 0 .1kg 1 d '1)3 (g 0 .1 k g 1)- (g 0.1kg'1)3

Years (Y) 1 0.004 1059.2" 975.7"

Reps, within Y 6 0.109** 35.2" 11.0

F, Crosses (C) 89 0.207" 152.8** 99.3"

GCA 9 1.018" 1113.4** 690.2"

SCA 35 0.161 76.3* 66.5"

Reciprocal (Rec.) 45 0.070" 28.3 20.3

C x Y 89 0.071** 31.4" 18.4" «- • vi 00 GCA x Y 9 0.084** 76.9"

SCA x Y 35 0.112" 37.0" 21.9"

Rec. x Y 45 0.033 17.1" 12.9

Midsilk' 1 0.782" 376.0** 0.7

GDDn 1 1,750" 73.4" 1321.8"

Error 532 0.035 10.2 12.2

Significant at 0.05 and 0.01 probability levels, respectively. ' Covariates. * Growing degree days. 92

Table 2.3. General combining ability (GCA) effects for first probe moisture (30DPM), second probe moisture (51DPM), and ear moisture loss rate (EMLR) from a 10-parent maize diallel cross grown at Baton Rouge, LA in 1992 and 1994.

Inbreds 30DPM 51DPM EMLR

- g 0 .1 k g 1 ------g 0.1kg 1 d 1 ......

L654 1.05 -1.23* 0.108**

L668 -1.62 -2.70** 0.051

LI 08 6.64** 3.89** 0.131**

L266 -1.39 -0.48 -0.044

L729 2.38* 2.18** 0.010

L329 5.19** 5.37** -0.008

L605 2.40** 0.99 0.067*

Hill -4.33** -3.36** -0.046

Mol 7 -5.37" -0.73 -0.221**

B73 -4.95** -3.93** -0.049

LSD,,,* 2.480 1.692 0.082

LSD,) in 3.562 2.431 0.118

*,** Significantly different from zero at 0.03 and 0.01 probability levels, respectively. 93

Table 2.4. Contrast sum of squares of first probe moisture (30DPM) (g 0.1kg2)2, second probe moisture (5IDPM) (g 0.1 kg2)2, and ear moisture loss rate (EMLR)(g 0.1kg 1 d'1)2 between L+ lines and out-of-state* lines.

Traits df Contrast sum of squares F value P value

30DPM 1 13.699 19.60 0.0017

51DPM 1 4.504 5.39 0.0454

EMLR 1 0.020 18.07 0.0021

t L108, L266, L329, L606, L654, L668, and L729. $ Hill. M o l7, and B73. 94

References

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A ten parent diallel was used to study the genetic natures of percent kernel

infection (PKI) by Aspergillus jlavus and ear moisture loss rate (EMLR) in maize.

General combining ability (GCA), specific combining ability (SCA), and

reciprocal mean squares for PKI were significant. The ratio 2 V f /(2Vg +VJ was

0.54. indicating that both GCA and SCA were important. The interaction mean

squares between year and GCA, SCA, and reciprocal were highly significant,

which implicit that more reliable information could be obtained by conducting

multi year experiments.

The GCA mean squares for EMLR, GCA and SCA mean squares for first

probe moisture (30DPM) and second probe moisture (51DPM) taken 30 and 51

days after midsilk, respectively, were significant, suggesting that EMLR, 30DPM,

and 5IDPM were genetically controlled traits. To select for low 51DPM, high

EMLR. positive GCA effects lor EMLR, and a relatively low 30DM were

important. The GCA effect was more inporlant than SCA effect for determining

EMLR. Extranuclear component(s) contributed to enhancement of EMLR. L668,

Hill, and B73 could be used as genetic sources in a maize breeding program for

lowering 5IDPM and gram moisture at harvest. A significant genetic correlation was detected between 51DPM and PKI. Low 5IDPM and low PKI could be achieved in a breeding program simultaneously. 97

Kernel infection by A. Jlovus and subsequent aflatoxin contamination is not

only a serious problem in agriculture, but also a difficult problem to solve. To

date, there is no single technique can eliminate this problem. Genetic studies have

shown that there is no complete resistant maize genotype available. However,

from this study we found several Ft crosses had significant low PKI than other

crosses and inbred lines L654 and L i08 had significant low PKI than most other

inbred lines. We may use these hybrids and/or inbred lines to build a population

for recurrent selection aiming at low PKI. In the future, plant breeders and plant geneticists need to screen more diversified maize germplasms from all over the

world. A promissing approach to solve this problem would be to find gene(s) coding the compound(s) which can kill the fungus or can inhibit the production of aflatoxins in the pathway of aflatoxin production. Molecular geneticists and

plant breeders should make more efforts to incorporate each other and solve the problem.

EMLR and ear moisture at physiological maturity (or 51DPM) are

important agronomic traits related to harvest ear moisture. More research should be done to determine the whole picture of EMLR from silking to harvest and estabiIish the relationships among EMLR, 5IDPM. harvest ear moisture, and yield in maize. VITA

Yudong Zhang was born in Yang Zhou, Jiangsu province of People’s

Rupublie of China on April 28, 1958, He graduated from Tai An High School in

July 1975 and then went back to his farm working for three years. In October

1978, he entered the Nanjing Agricultural College (now called Nanjing

Agricultural University). In July 1982, he graduated with a Bachelor of Science degree in Agronomy from the Nanjing Agricultural University. The author entered graduate school in September 1982 at the Nanjing Agricultural University and received a Master of Science degree in Agronomy from the University in

December 1985. From January 1986 to June 1990. the author worked at Nanjing

Agriculture & Techology College as a lecturer and a plant breeder. From July

1990 to Feburary 1991. He worked at the Slate Science & Technology Committee in Beijing. Then, he accepted a fellowship from Netherlands Fellowships

Programma and went to the International Agricultural Center in Netherlands for training in Applied Plant Breeding for three and half months. After training, he went back to the Nanjing Agriculture & Technology College and worked as a lecturer and a plant breeder. In June 1992. the author accepted a graduate assistantship from the Louisiana Slate University and now is a candidate for the

Ph.D. degree in Agronomy (Crop Science).

98 DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Yudong Zhang

Major Field: Agronomy

Title of Dissertation: Diallel Analyses of Percent Kernel Infection by Aspergillus Flavua and Ear Moisture Loss Rate in Maize

Approved:

Major Pf6fess6rand Chdffnian '

Dean'of the '''Graduate School

EXAMINING COMMITTEE:

j? "LdJL-

Date of Examination:

November 6 . 1995