Use of Classical and Molecular Technologies for Developing Aflatoxin Resistance in Crops - Agricultural Research Service, Southern Regional Research Ctr

Goals / Objectives
Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance-associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus-host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms.

Project Methods
Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over-expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally.

Progress 06/14/16 to 04/18/21

Outputs
PROGRESS REPORT Objectives (from AD-416): Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance- associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus- host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms. Approach (from AD-416): Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over- expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally. This is the final report for the Project 6054-42000-025-00D terminated in April 2021, which has been replaced by new Project 6054-42000-027-00D. For additional information, see the new project report. Significant progress has been made by ARS scientists in New Orleans, Louisiana in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. One of the best means of combating aflatoxin contamination is through development of resistant corn lines through classical or molecular breeding. Objective 1, ARS researchers in New Orleans, Louisiana, have identified several proteins in corn kernels linked with enhanced resistance to infection by the aflatoxin (AF, a carcinogen)-producing fungus, Aspergillus (A.) flavus. These genes have been transferred into commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, ARS researchers produced six corn varieties that were deposited into the ARS repository and made available to other researchers. These six lines were used to develop new elite hybrids with AF resistance in several African countries and in the United States. They are available to all domestic and international breeders. Objective 2, ARS researchers in New Orleans, Louisiana, in collaboration with the J. Craig Venter Institute, La Jolla, California, used modern RNA- Sequencing (RNA-seq, a means of determining levels of activity of individual genes in both the fungus and corn) technology to study the expression of genes during the corn-A. flavus interaction. For this work, two resistant and one susceptible corn lines were evaluated. Comparative analysis indicated several novel corn genes and biological processes related to resistance to fungal infection and AF contamination. To further identify novel genes of interest, ARS scientists incorporated genomic and field data from a genome wide association analysis (GWAS) of corn varieties with gene expression data. These efforts identified significant association between flavonoid (plant nutrients with strong antioxidant and defense capabilities) genes and AF contamination. One of the resistant lines has a higher production rate of the flavonoid metabolite called naringenin than susceptible line suggesting the important roles flavones play in fungal growth and AF production. Non-coding small RNAs (ncRNAs, do not encode for proteins) present in corn lines were also sequenced and analyzed by ARS scientists in New Orleans, Louisiana in collaboration with researchers at Louisiana State University, Baton Rouge, Louisiana to identify microRNAs (miRNAs, a class of ncRNAs that can control the expression of specific genes) with potential roles in A. flavus resistance. Differences in miRNAs were observed between resistant and susceptible corn lines. In summary, candidate genes and small RNAs with positive roles in corn resistance against A. flavus have been identified. The value of these genes in marker-assisted breeding will be further investigated. Using the information from Objectives 1 and 2 and from the sibling project 6054-41420-008-00D ARS researchers in New Orleans, Louisiana, continued to make significant progress in molecular breeding of corn for resistance to A. flavus and AF contamination under Objective 3. a) Transgenic corn kernels expressing a synthetic peptide gene demonstrated a significant reduction in fungal growth and AF contamination reduction. Field evaluation of these corn lines in 2021 was discontinued because of excessive heat and rain in the test site. Corn was also transformed with a gene from another plant that encodes a novel antifungal protein. This antifungal protein inhibits a key enzyme (alpha-amylase) that is necessary for the fungus to grow and infect seed. (b) To understand the contribution of a corn kernel protein to A. flavus resistance, it was silenced using a ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) approach in transgenic corn lines. This gene also affected the functioning of other genes involved in disease resistance. Similar RNAi- based approaches were carried out to silence fungal genes that are critical for the fungus to grow, infect and produce toxins. Several corn lines capable of shutting down fungal genes that control growth such as a- amylase and p2c (pectinase), AF biosynthetic genes or global regulators such as aflR, aflS, aflM, aflC, veA and nsdC showed significant AF reduction in transgenic kernels. Two of these lines (aflM and p2c) also showed significant resistance to AF contamination under field conditions. A significant reduction (up to 98%) was observed of AFs in select transgenic corn lines expressing foreign genes or that are capable of silencing fungal genes. Additional corn transformation experiments were not possible due to shut down of service provides during pandemics. (c) Unlike in corn, no resistance to A. flavus has been identified in cotton seed sock so far. ARS scientists in New Orleans, Louisiana, in collaboration with scientists at the University of Louisiana-Lafayette assayed several varieties of cotton to identify natural resistance to A. flavus and AF accumulation. Upon screening for any innate resistance to the fungus, old-world cotton varieties of Gossypium (G.) arboreum were found to be most resistant and commercially cultivated upland cotton varieties were all susceptible to A. flavus infection. (d) Fatty acid accumulation in developing cottonseed was well-correlated with the ability of an AF-producing fungal strain to grow and produce AFs. This study identified key factors controlling A. flavus infection and AF production in cottonseed. (e) Transgenic cotton lines expressing an antifungal synthetic peptide designated D4E1 demonstrated resistance to A. flavus in greenhouse studies or seedling pathogens under field conditions. They were field-tested by ARS scientists in collaboration with University of Arizona for resistance to AF contamination. Results from the small field experiment were inconclusive due to lack of natural A. flavus infection and AF contamination. (f) New experiments to produce transgenic corn lines expressing improved synthetic peptides with potency against A. flavus have also been initiated by ARS researchers in New Orleans, Louisiana under a cooperative agreement. ARS researchers, in collaboration with Louisiana State University, Baton Rouge, Louisiana, also identified a key gene that is overexpressed in cotton boll pericarp (the outer wall) called spot11 catalase from RNA-seq analyses (Objective 2). Due to pandemics, limited number of transgenic cotton lines expressing this gene were regenerated for further analyses on resistance to fungal infection and AF contamination. Several cultures were also lost during maximized telework. (g) ARS researchers have also demonstrated the critical roles in fungal growth and toxin production by polyamines (PAs), which are ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (drought, salt) stress. First, inactivation of a key fungal gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. In addition, analysis of maize genotypes susceptible or resistant to Aspergillus flavus identified a key gene (S-adenosylmethionine decarboxylase or SAMDC) that regulates the production of higher PAs, possibly contributing to fungal resistance. The fungal and plant genes identified from this work provide potential targets for improvement of maize resistance to fungal colonization and aflatoxin production. In addition to developing crops resistant to AF contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University (MSU) in Mississippi State, Mississippi, based at the Stennis Space Center, Mississippi, developed a non-invasive, inexpensive and rapid imaging technique that collects and processes information from across the light-spectrum under Objective 4 to detect and quantify AFs in corn kernels. These special cameras have already demonstrated the ability to differentiate toxigenic and atoxigenic Aspergillus flavus strains. A patented method to analyze different wavelengths was initially used. Subsequently, a joint effort between ARS researchers in New Orleans, Louisiana, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel rapid method to improve AF detection by a dual-camera imaging system. This system was successfully tested with fungal- inoculated commercial corn in the United States. With support from the United States Agency for International Development (USAID), ARS researchers and collaborators perfected the concept into a low-cost portable device called AflaGoggles to detect AF contamination in corn kernels for use in developing countries. The device has now been updated to a tablet-based system equipped with UV-LED light source. The device employs an in-house developed Android App for image acquisition and detection of contaminated kernels for manual removal by the user. Following successful experiments in the United States on corn kernels, work is being continued to improve the accuracy of detection and the device⿿s portability for field deployment. Record of Any Impact of Maximized Teleworking Requirement: Due to the maximized telework during the recent pandemic posture in FY2021 neither ARS researchers at New Orleans, Louisiana, nor our collaborators or service providers (also locked out of their workplaces or pandemics-forced closures) have been able to conduct research or make progress on three of the project⿿s four objectives that involves extensive laboratory, greenhouse, field, and service-related contract work. Several cotton and corn cell cultures from in-house transformation experiments, that were initiated by ARS scientists in New Orleans, Louisiana 15-20 months ago, were lost during this period and it has significantly delayed the completion of transgenic experiments to fully meet the research goals and milestones. Field evaluation by ARS scientists in New Orleans, Louisiana of transgenic corn lines was not feasible due to unfavorable weather conditions such as excessive heat, heavy rains, and freezing temperatures until early April 2021. ACCOMPLISHMENTS 01 Corn plants shut down fungal genes and toxin production. Corn is an important food and feed crop, and it is highly susceptible to A. flavus infection and aflatoxin (a carcinogen) contamination. Transgenic corn plants were generated by ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, to selectively shut down one of the fungal genes (called p2c-polygalacturonase or pectinase) necessary for its growth, infection and spread. The transgenic corn lines with the capacity to silence p2c were tested in the laboratory and field for three years. Aflatoxin production was significantly reduced (60- 95%) corresponding to reduced levels of fungal growth (up to 40%). This technology, made possible by the short-lived silencing RNA molecules, does not require expression of a foreign protein in the plant so food produced from resistant transgenic lines of corn should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will also serve as an excellent parent material to transfer the resistance trait to other commercial varieties. Aflatoxin-resistant corn lines improve food safety and security while minimizing economic losses borne by farmers. 02 Bacteria naturally found in corn kernels are associated with reduced toxin levels. ARS scientists in New Orleans, Louisiana, suggest a toxin, called aflatoxin, produced by the fungus Aspergillus (A.) flavus during growth on corn is a worldwide food safety problem. Aflatoxins are potent carcinogens that adversely impact human and animal health and contamination of crops with aflatoxins costing stakeholders tens of millions of dollars annually. Management of aflatoxin contamination in corn is complex so identification of any factors that contribute to kernel resistance is of great importance in developing control strategies. Using sophisticated DNA sequencing technologies, ARS researchers in New Orleans, Louisiana were able to identify specific groups of bacteria that were present in greater numbers in kernels of resistant corn lines compared to susceptible lines. Many of the identified bacteria are known to produce compounds that can stop the growth of toxin-producing fungi. Continued evaluation by ARS scientists in New Orleans, Louisiana of bacteria predicted to possess antifungal and anti-aflatoxigenic properties will aid in their development as effective agents for enhanced resistance to A. flavus infection and aflatoxin contamination thus ensuring a safer and more secure food supply.

Impacts
(N/A)

Publications

Tao, F., Yao, H., Hruska, Z., Kincaid, R., Rajasekaran, K., Bhatnagar, D. 2020. A novel hyperspectral-based approach for identification of maize kernels infected with diverse Aspergillus flavus fungi. Biosystems Engineering. 200:415-430. https://doi.org/10.1016/j.biosystemseng.2020.10. 017.
Majumdar, R., Kandel, S.L., Cary, J.W., Rajasekaran, K. 2021. Changes in bacterial endophyte community following aspergillus flavus infection in resistant and susceptible maize kernels. International Journal of Molecular Sciences. 22(7). Article 3747. https://doi.org/10.3390/ ijms22073747.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance- associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus- host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms. Approach (from AD-416): Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over- expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally. Substantial progress has been made in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Objective 1, ARS researchers in New Orleans, Louisiana, have identified several native proteins in corn kernels linked with enhanced resistance to infection by the aflatoxin (AF, a carcinogen)-producing fungus, Aspergillus (A.) flavus. Genes encoding such resistant-proteins have been transferred into commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, ARS reseachers at New Orleans, Louisiana, produced six inbred lines (with less genetic variation) that were deposited into the corn germplasm or repository and made available to other researchers. The combining ability of exotic corn lines resistant to AF accumulation was examined as a means to generate new inbred lines for cultivation in the United States with enhanced resistance to AF contamination. Two productive corn varieties with resistance to AF contamination were released as ⿿Sammaz45⿝ in Nigeria and ⿿Syn2-YF2⿝ in Cameroon. Several elite hybrids formed from AF resistant inbred lines are currently in advanced stage of testing for possible cultivation and they are available to all breeders from the ARS corn germplasm. Meanwhile, detailed analyses of the proteins contributing to resistance to toxin-producing A. flavus in these inbreds and hybrids are being conducted at the Southern Regional Research Center (SRRC), New Orleans, Louisiana. ARS researchers in New Orleans, Louisiana, have also demonstrated the critical roles in fungal growth and toxin production of polyamines (PAs), ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (environmental) stress. ARS scientists in New Orleans, Louisiana, have initiated a novel project based on engineering for PA content by introducing a native key PA biosynthetic gene (S-adenosylmethionine decarboxylase or SAMDC) into corn kernels to protect from AF contamination (Objective 3). Under Objective 2, ARS researchers in New Orleans, Louisiana, in collaboration with the J. Craig Venter Institute, La Jolla, California, used modern RNA-Sequencing (RNA-seq, a means of determining levels of activity of individual genes in both the fungus and corn) technology to study the expression of genes during the corn-A. flavus interaction. Comparative analysis indicated several novel gene candidates and biological processes related to host resistance to fungal infection and AF contamination. The genes and gene pathways of interest identified from the RNA-seq study include tetraspanins, acetylornithine deacetylase, macrophage migratory inhibitory factor, and genes in the flavonoid and flavone biosynthetic pathway and protein detoxification. Gene co- expression analysis identified at least two gene network modules that were highly preserved in resistant lines compared to susceptible corn line, indicating their role(s) in resistance mechanisms. For example, genes for jasmonic acid signaling, nitrate assimilation, and production of glucoside and long chain fatty acid were highly enriched in these modules. Non-coding small RNAs (ncRNAs, do not encode for proteins) present in corn lines were also sequenced and analyzed in collaboration with researchers at Louisiana State University, Baton Rouge, Louisiana to identify microRNAs (miRNAs, a class of ncRNAs that can control the expression of specific genes) with potential roles in A. flavus resistance. Differences in miRNAs were observed between resistant and susceptible corn lines. Expression analysis of the candidate target genes of such miRNAs and potential novel miRNAs are being carried out. These data will be compared with the RNA-seq data (presented above) to identify the miRNA-mRNA modules demonstrating roles in resistance to A. flavus infection in corn. Analysis of A. flavus small RNA (smRNA) sequence data by ARS researchers in New Orleans, Louisiana, indicated the presence of unconventionally sized (approx. 15 base pairs, normally 21-24 base pairs) smRNA molecules. These inhibitory smRNAs were generated by three potential Dicer-like proteins (Dicer is an enzyme which facilitates the activation of the RNA- induced silencing complex or RISC, which is essential for targeted gene silencing). A series of A. flavus Dicer mutant strains have been generated where one, two, or all three of these putative Dicer-like genes were knocked out in an effort to determine which of these genes is responsible for production of these unusually small RNAs. Objective 3, ARS researchers in New Orleans, Louisiana, continued to make significant progress in the genetic engineering of corn for resistance to A. flavus and AF contamination. Transgenic corn lines expressing double- stranded RNA molecules in corn seed were analyzed that specifically target fungal toxin biosynthetic genes for silencing. Recently, corn plants with the capability to silence a key enzyme involved in the AF biosynthetic pathway (aflM) were field tested for three years and were found to provide significant resistance to natural AF contamination (up to 95% reduction). ARS researchers in New Orleans, Louisiana, are also generating more corn lines in which up to four fungal genes required for AF production (for example, aflR, aflS, aflM, aflC, veA, nsdC) have been targeted for silencing. These transgenic corn lines will be analyzed further for their effectiveness in controlling fungal growth and toxin production. New experiments to produce transgenic corn lines expressing improved synthetic peptides have also been initiated under a cooperative agreement. Similarly, ARS researchers New Orleans, Louisiana, in collaboration with Louisiana State University, Baton Rouge, Louisiana, identified a key gene that is overexpressed in cotton boll pericarp (the outer wall) called spot11 catalase from RNA-seq analyses (Objective 2). Limited number of transgenic cotton lines expressing this gene have been regenerated for further molecular analyses and resistance to fungal infection and AF contamination. In addition to developing crops resistant to AF contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University (MSU) in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral imaging technique (that employs a large part of the electromagnetic spectrum, especially that includes those parts of the spectrum invisible to the eye) that collects and processes information from across the light-spectrum. This imaging technique detects and quantifies aflatoxins in corn kernels under Objective 4. Hyperspectral instruments have already demonstrated the ability to differentiate AF and non-AF A. flavus strains. A spectral signature to detect AF-contaminated corn has been developed and licensed. A joint effort between ARS researchers, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel method to improve AF detection utilizing the accuracy of multispectral imaging technology and to develop commercial, rapid, screening equipment for AF-contaminated corn. With support from the United States Agency for International Development, ARS researchers and collaborators upgraded the initial AflaGoggles concept into a low-cost portable technology to detect AF contamination in corn kernels for use in developing countries. The current outcome is a portable tablet-based detection device equipped with UV-LED light source. The device employs an Android App developed in-house for image acquisition, detection, and marks the contaminated kernels so the user can manually sort them out. Experiments have been implemented using inoculated corn kernels, which mimics natural field AF contamination. The research team also continues to seek support from other sources to implement field tests in developing countries where AF contamination of crops is endemic. Accomplishments 01 Classic breeding results in aflatoxin resistant corn varieties. Aflatoxin (a carcinogen) contamination in a major food crop worldwide, corn, caused by Aspergillus (A.) flavus is not only a food safety problem impacting human and animal health but also it results in huge economic losses to farmers and stakeholders due to contaminated produce being unsuitable for market. In order to develop strategies to mitigate aflatoxin contamination of corn and other crops, it is important to develop corn lines resistant to A. flavus infection and aflatoxin contamination in edible kernels. Resistant proteins and their corresponding genes have been identified in corn lines by ARS researchers in New Orleans, Louisiana, and these genes have been transferred to commercial varieties by classical breeding and they are available to U.S. breeders. In collaboration with the International Institute of Tropical Agriculture, Nigeria, additional hybrids were developed which showed resistance to aflatoxin contamination. Two productive corn varieties with resistance to aflatoxin contamination were released as ⿿Sammaz45⿝ in Nigeria and ⿿Syn2-YF2⿝ in Cameroon for cultivation. Additional hybrids for other countries where aflatoxin contamination is endemic are also being developed. Aflatoxin-resistant corn lines improve food safety and security worldwide while reducing economic losses borne by farmers. 02 Host-induced gene silencing of Aspergillus (A.) flavus genes reduces infection and aflatoxin production in corn kernels. Corn is an important food and feed crop and it is highly susceptible to A. flavus infection and aflatoxin (a carcinogen) contamination. Transgenic corn plants were generated by ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, to selectively silence one of the key aflatoxin pathway genes called aflM using a host-induced gene silencing approach. The transgenic lines with the capability of silencing fungal aflM were field tested for three years. Aflatoxin production was significantly reduced (up to 95%) under natural infection and even under artificial infection the aflatoxin levels were reduced by 77%. This technology, made possible by the short-lived silencing RNA molecules, does not require expression of a foreign protein in the plant so food produced from resistant transgenic lines of corn should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will also serve as an excellent parent material to transfer the resistance trait to other commercial varieties. Aflatoxin-resistant corn lines improve food safety and security while minimizing economic losses borne by farmers.

Impacts
(N/A)

Publications

Tao, F., Yao, H., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. Detection of aflatoxin B1 on corn kernel surfaces using visible-near infrared spectroscopy. Journal of Near Infrared Spectroscopy. 28(2):59-69.
O'Neill, M.J., Chan, K., Jaynes, J.M., Knotts, Z., Xu, X., Abisoye- Ogunniyan, A., Guerin, T., Schlomer, J., Li, D., Cary, J.W., Rajasekaran, K., Yates, C., Kozlov, S., Andresson, T., Rudloff, U. 2020. LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 181:113093.
Raruang, Y., Omolehin, O., Hu, D., Wei, Q., Han, Z.-Q., Rajasekaran, K., Cary, J.W., Wang, K., Chen, Z.-Y. 2020. Host induced gene silencing targeting Aspergillus flavus aflM reduced aflatoxin contamination in transgenic maize under field conditions. Frontiers in Microbiology. 11:754.
Sengupta, S., Rajasekaran, K., Baisakh, N. 2019. Basic characterization of plant actin depolymerizing factors: a simplified, streamlined guide. Protocol Exchange.
Hruska, Z., Yao, H., Kincaid, R., Tao, F., Brown, R.L., Cleveland, T.E., Rajasekaran, K., Bhatnagar, D. 2020. Spectral-based screening approach evaluating two specific maize lines with divergent resistance to invasion by aflatoxigenic fungi. Frontiers in Microbiology. 10:3152.
Meseka, S., Williams, W.P., Warburton, M.L., Brown, R.L., Ortega, A., Bandyopadhyay, R., Menkir, A. 2018. Heterotic affinity and combining ability of exotic maize inbred lines for resistance to aflatoxin accumulation. Euphytica. 214:184.
Musungu, B.M., Bhatnagar, D., Payne, G.A., O'Brian, G., Quiniou, S., Geisler, M., Fakhoury, A. 2020. Use of dual RNA-seq for systems biology analysis of zea mays and Aspergillus flavus interaction. Frontiers in Microbiology. 11:853.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance- associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus- host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms. Approach (from AD-416): Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over- expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally. Substantial progress has been made in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Under Objective 1, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, continue to identify several native proteins in corn kernels with enhanced resistance to infection by the aflatoxin producing fungus, Aspergillus (A.) flavus. Such resistance-associated proteins and their corresponding genes have been identified in corn lines and these genes have been transferred to commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, ARS researchers produced six corn lines named TZAR 101-106 that showed resistance not only to aflatoxin-producing fungi but also to a Fusarium fungus that produces another toxin called fumonisin. Limited field evaluations of these six corn lines and test-crosses with domestic lines have been conducted as a part of Southeast Regional Aflatoxin Test (SERAT2) in multiple locations (Starkville, Mississippi; College Station, Texas; Lubbock, Texas; and Carbondale, Illinois). TZAR lines and test-crosses performed well against aflatoxin contamination. Meanwhile, detailed analyses of the proteins (proteomic analysis) contributing to resistance to toxin-producing fungi in TZAR lines and the test-crosses are being conducted at the Southern Regional Research Center (SRRC), New Orleans, Louisiana. ARS researchers in New Orleans, Louisiana, have also demonstrated the critical roles in fungal growth and toxin production by polyamines (PAs), which are ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (drought, salt) stress. First, inactivation of a key fungal gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. In addition, analysis of maize genotypes susceptible or resistant to Aspergillus flavus identified a key gene (S-adenosylmethionine decarboxylase or SAMDC) that regulates the production of higher PAs, possibly contributing to fungal resistance. The fungal and plant genes identified from this work provide potential targets for improvement of maize resistance to fungal colonization and aflatoxin production. Under Objective 2, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, conducted experiments on a genome-wide transcriptome (the sum of all the actively expressed genes of a corn plant) analysis of the corn-Aspergillus (A.) flavus interaction. In collaboration with the J. Craig Venter Institute, La Jolla, California, ARS researchers used the modern ribonucleic acid-sequencing (RNA-Seq; a means of determining levels of activity of individual genes in both the fungus and corn) technique to study the expression of genes during the corn-A. flavus interaction. Comparative analysis will be made of A. flavus-infected kernels of the aflatoxin and drought resistant hybrid TZAR 102, released by ARS, along with an aflatoxin-resistant line (MI82) and a susceptible control (Va35). This will allow us to delineate the molecular genetic differences that might explain the enhanced resistance to A. flavus. Big data analysis of ribonucleic acid (RNA) sequencing is on-going with Louisiana State University, Baton Rouge, Louisiana, to identify key genes at the mRNA (transmit genetic information from DNA to make amino acid sequence of the protein) and microRNA (miRNA - small non-coding RNA molecule containing about 22 nucleotides that functions in RNA silencing and regulation of gene expression) levels. An interactome analysis which analyzes the whole set of molecular interactions in a particular cell based on the RNA-Seq data of the A. flavus-corn interaction is also being performed to enable the identification of key global regulators of A. flavus growth and aflatoxin biosynthesis as well as developmental and virulence (ability to cause infection) factors that can serve as targets for intervention strategies. This analysis will shed light specifically on the mechanisms of fungal pathogenesis and corn resistance. Using a similar approach ARS researchers in collaboration with Louisiana State University, Baton Rouge, Louisiana, identified a key gene that is overexpressed in cotton boll pericarp (the outer wall) called spot11 catalase. Transgenic cotton lines expressing this gene are being regenerated and will be analyzed for molecular and phenotypic resistance to fungus. Under Objective 3, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, made significant progress in the genetic engineering of corn for resistance to Aspergillus (A.) flavus and aflatoxin production. ARS researchers analyzed transgenic maize lines expressing double-stranded ribonucleic acid (RNA) molecules from maize seed that target two specific A. flavus genes, either alone or in combination. These two target genes, nsdC and veA, which are required by A. flavus for production of aflatoxin and fungal survival structures, sclerotia, along with numerous other toxic secondary metabolites. An in vitro maize seed infection assay with A. flavus showed up to 75% reduction of fungal growth in the infected ribonucleic acid interference (RNAi) seeds targeting nsdC and veA as compared to the seeds from a control plant. A significant 86% reduction of aflatoxins was observed along with 80% reduction of another important fungal secondary metabolite called cyclopiazonic acid (CPA). ARS researchers in New Orleans, Louisiana, have also generated more corn lines in which up to four fungal toxin biosynthetic genes or global regulators (aflR, aflS, aflM, aflC, veA, nsdC) have been targeted for silencing. These corn lines will be analyzed further for their effectiveness in controlling fungal growth and toxin production. In addition to developing crops resistant to aflatoxin contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University (MSU) in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral imaging technique that collects and processes information from across the light-spectrum. This imaging technique detects and quantifies aflatoxins in corn kernels under Objective 4. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic Aspergillus flavus strains. A spectral signature to detect aflatoxin-contaminated corn has been developed and licensed. A hyperspectral camera system based on pushbroom and rotational scan for whole corn ear surface imaging and contamination mapping has been developed and tested. A joint effort between ARS researchers, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel method to improve aflatoxin detection utilizing accuracy of multispectral imaging technology and to develop commercial, rapid, screening equipment for aflatoxin-contaminated corn. For this effort, a prototype dual-camera based multispectral imaging system for the purpose of rapidly screening aflatoxin contamination has been designed, developed, and evaluated. Funds were also secured from the National Science Foundation (2017-2018) for a project on ⿿Novel method to improve aflatoxin detection accuracy of multispectral imaging technology. ⿝ The dual-camera system has been tested with field-inoculated corn as well as commercial samples from three states, Arkansas, Mississippi, and Tennessee, in 2018. With support from the United States Agency for International Development, ARS researchers and collaborators developed a low-cost portable technology to detect aflatoxin contamination in corn kernels for use in developing countries. The current outcome is a tablet- based sorting device equipped with UV-LED light source. The research team has secured funds from LaunchTN in Nashville, Tennessee, and is actively seeking support from other sources to implement field tests in developing countries. Accomplishments 01 Host-induced gene silencing of Aspergillus (A.) flavus genes to reduce infection and aflatoxin production in maize kernels. Maize is an important food and feed crop and it is highly susceptible to Aspergillus (A.) flavus infection and aflatoxin (AF, a carcinogen) contamination. ARS researchers in New Orleans, Louisiana, have produced transgenic maize plants, which can withstand fungal infection and toxin production through ribonucleic acid (RNA) interference-mediated host- induced gene silencing (HIGS; a mechanism in the plant that produces small RNA molecules that can target specific genes for silencing). The HIGS process targets key A. flavus genes to reduce fungal virulence, growth and/or AF biosynthesis in susceptible maize crop and it has proven to be a promising and consumer-friendly approach to control dangerous levels of carcinogenic AF. The genes that were targeted for silencing include the veA and nsdC genes, both global regulators required for normal A. flavus development and AF production. Fungal growth was monitored in transgenic kernels using a green fluorescent protein (GFP)-expressing A. flavus and AF measurement was carried out by UPLC (Ultra High Pressure Liquid Chromatography). Results from transgenic kernel infection studies demonstrated significant reductions in fungal growth, invasion, and AF production in transgenic maize ribonucleic acid interference lines (85-90% reduction compared to controls). A significant reduction by up to 82% was also observed in another toxic secondary metabolite called cyclopiazonic acid (CPA). 02 Transgenic maize kernels expressing a synthetic peptide are resistant to aflatoxin contamination. Maize is an important food and feed crop and it is highly susceptible to Aspergillus flavus infection and aflatoxin (a carcinogen) contamination. ARS researchers in New Orleans, Louisiana, have determined that a synthetic peptide (small protein) called AGM 182, modeled after an antimicrobial peptide from horseshoe crab, provides significant control of Aspergillus flavus growth and toxin production. ARS researchers also determined this peptide is not toxic to animals and human beings. Genetically altered corn kernels expressing the AGM 182 gene demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). The development of corn containing the peptide was a collaborative effort between ARS researchers and a CRADA partner that provided the synthetic peptide, along with a scientist at University of Arkansas, Fayetteville, Arkansas. Transgenic lines will be advanced to at least two more progenies and multiplied in the greenhouse for evaluation under field conditions. 03 Maize genotypes resistant to aflatoxin contamination have higher levels of polyamines than susceptible lines. Maize, an important food and feed crop, is highly susceptible to Aspergillus flavus infection. Upon infection, the fungus produces carcinogenic aflatoxins and numerous other toxic secondary metabolites that adversely affect crop value and human health worldwide. The role of polyamines (PAs), which are ubiquitous nitrogenous molecules that act as global regulators of growth and development fortifying plant productivity under ambient or stress conditions, in Aspergillus (A.) flavus resistance and aflatoxin production in resistant and susceptible maize lines was determined. In collaboration betweem ARS researchers in New Orleans, Louisiana, scientists from USDA Forest Service and University of New Hampshire, Durham, New Hampshire, analysis of PA content in both resistant (TZAR102 and MI82) and susceptible (SC212) corn kernels was conducted. The analysis indicated that resistant varieties showed higher expression of PA biosynthetic genes upon A. flavus infection compared to the susceptible control kernels. The resistant lines accumulated higher amounts of PAs such as spermidine (Spd), spermine (Spm), and specific antimicrobial PA conjugates, showed altered amino acids content, and had lower levels of fungal load and aflatoxin contamination. In a parallel study with A. flavus, inactivation of a key PA gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. Combined together, these results provide a valid means of controlling A. flavus growth and aflatoxin production in corn through engineering of PA biosynthesis, either by over expression in the plant or by silencing the fungal gene by host-induced gene silencing.

Impacts
(N/A)

Publications

Tao, F., Yao, H., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. Use of visible-near-infrared (Vis/NIR) spectroscopy to detect aflatoxin B1 on peanut kernels. Applied Spectroscopy. 73(4):415-423.
Rajasekaran, K., Sayler, R.J., Majumdar, R., Sickler, C.M., Cary, J.W. 2019. Inhibition of Aspergillus flavus growth and aflatoxin production in transgenic maize expresing the a-amylase inhibitor from Lablab purpureus L. Journal of Visualized Experiments. 144:e59169.
Sengupta, S., Rajasekaran, K., Baisakh, N. 2018. Natural and targeted isovariants of the rice actin depolymerizing factor 2 can alter its functional and regulatory binding properties. Biochemical and Biophysical Research Communications. 503:1516-1523.
Moore, J., Rajasekaran, K., Cary, J.W., Chlan, C. 2018. Mode of action of the antimicrobial peptide D4E1 on Aspergillus flavus. International Journal of Peptide Research and Therapeutics. 25(3):1135-1145.
Majumdar, R., Minocha, R., Lebar, M.D., Rajasekaran, K., Long, S., Carter- Wientjes, C.H., Minocha, S., Cary, J.W. 2019. Contribution of maize polyamine and amino acid metabolism toward resistance against Aspergillus flavus infection and aflatoxin production. Frontiers in Plant Science. 10:692.
Han, D., Yao, H., Hruska, Z., Kincaid, R., Rajasekaran, K., Bhatnagar, D. 2019. Development of high-speed dual-camera system for batch screening of aflatoxin contamination of corn using multispectral fluorescence imaging. Transactions of the ASABE. 62(2):381-391.
Hruska, Z., Yao, H., Kincaid, R., Brown, R.L., Bhatnagar, D., Cleveland, T. E. 2017. Temporal effects on internal fluorescence emissions associated with aflatoxin contamination from corn kernel cross-sections inoculated with toxigenic and atoxigenic Aspergillus flavus. Frontiers in Microbiology. 8:1718.
Xing, F., Yao, H., Liu, Y., Dai, X., Brown, R.L., Bhatnagar, D. 2017. Recent developments and applications of hyperspectral imaging for rapid detection of mycotoxins and mycotoxigenic fungi in food products. Critical Reviews in Food Science and Nutrition. 59(1):173-180.
Tao, F., Yao, H., Zhu, F., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. A rapid and nondestructive method for simultaneous determination of aflatoxigenic fungus and aflatoxin contamination on corn kernels. Journal of Agricultural and Food Chemistry. 67:5230-5239.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance- associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus- host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms. Approach (from AD-416): Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over- expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally. Substantial progress has been made in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Under Objective 1, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, previously identified several native proteins in corn kernels that resist infection by the fungus, Aspergillus (A.) flavus that produces aflatoxins. Such resistance- associated proteins and their corresponding genes have been identified in corn lines and these genes have been transferred to commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, six corn lines named TZAR 101-106 were developed and they showed resistance not only to aflatoxin-producing fungi but also to a Fusarium fungus that produces another toxin called fumonisin. Limited field evaluations of these six corn lines and test- crosses with domestic lines have been planned and will be conducted in the coming years. Meanwhile, detailed analysis of the proteins (proteomic analysis) contributing to the resistance to the toxin-producing fungi is being conducted at the Southern Regional Research Center (SRRC). Under Objective 2, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, conducted experiments on a genome-wide transcriptome (the sum of all the actively expressed genes of a corn plant) analysis of the corn-Aspergillus (A.) flavus interaction. In collaboration with the J. Craig Venter Institute, La Jolla, California, ARS researchers used the modern ribonucleic acid-sequencing (RNA-Seq; a means of determining levels of activity of individual genes in both the fungus and corn) technique to study expression of genes during the corn-A. flavus interaction. Comparative analysis will be made of A. flavus- infected kernels of hybrid TZAR 102, resistant to aflatoxin contamination and drought, released by ARS, along with a resistant line (MI 82) and a susceptible check (Va35) to delineate the molecular genetic differences that might explain the enhanced resistance to A. flavus. Data generated from RNA sequencing is currently being analyzed in-house. An interactome (the whole set of molecular interactions in a particular cell) analysis based on the RNA-Seq data will also be performed enabling identification of key global regulators of A. flavus growth and aflatoxin biosynthesis as well as developmental and virulence (ability to cause infection) factors that can serve as targets for intervention strategies. This analysis will shed light specifically on the mechanisms of fungal pathogenesis and corn resistance. A similar genome-wide transcriptome profiling study has already been completed in cotton where ARS researchers identified several key genes that were turned on during infection of A. flavus in cottonseed. Recently, a comparative transcriptome analysis was also performed in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, that identified common genes that were significantly differentially expressed in cotton, corn, and peanut in response to A. flavus. ARS researchers and collaborators identified 26 genes common across all three crops that were considered candidate A. flavus resistant genes, which could be used to improve resistance to aflatoxin production in susceptible crops. Under Objective 3, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, made significant progress in genetic engineering of corn and cotton for resistance to Aspergillus (A.) flavus and aflatoxin production. a) Transgenic corn kernels expressing a synthetic peptide (small protein) gene AGM 182 (modeled after an antimicrobial peptide from horseshoe crab) demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). ARS researchers obtained the synthetic peptide from Nexion, Inc. and a scientist at University of Arkansas, Fayetteville, Arkansas, collaborated on transformation of corn. b) In another experiment in collaboration with scientists at the University of Arkansas in Fayetteville, Arkansas, reduction in fungal growth and toxin production was also observed from sixth generation transgenic corn kernels expressing a gene from another plant, hyacinth beans, that encodes a novel antifungal seed protein. This antifungal protein inhibits a key enzyme (alpha-amylase) that is necessary for the fungus to grow and infect seed. Significantly, reduced fungal growth and toxin production (up to 88%) was observed with transgenic corn kernels expressing this natural protein. c) Significant progress has also been achieved in experiments to understand the contribution of native corn kernel proteins to A. flavus resistance. One such protein is called Pathogenesis�Related maize seed protein (PRms). To understand its role in fungal resistance, the gene was first silenced using a ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) approach in transgenic corn lines. Down-regulation of PRms in transgenic kernels resulted in a ~250-350% increase in Aspergillus flavus growth accompanied by a 4.5 to 7.5-fold higher accumulation of aflatoxins than the control plants, confirming its central role in fungal resistance. This particular gene also affected the functioning of other genes associated with disease resistance. d) Other RNAi-based approaches were carried out to silence fungal genes as well that are critical for the fungus to grow, infect and produce toxins. One such experiment to target and silence a fungal gene that codes for alpha-amylase (essential for fungal growth) resulted in development of transgenic corn lines capable of resisting Aspergillus flavus growth and aflatoxin production. The transgenic corn plants not only reduced the fungal amylase gene expression resulting in decreased fungal growth but also significantly reduced aflatoxin production by 98% in kernels. e) ARS researchers analyzed transgenic maize lines expressing RNAi in maize seed only that target two specific Aspergillus flavus genes- nsdC and veA which are required by A. flavus for production of aflatoxin and sclerotia [fungal survival structures], and numerous other toxic secondary metabolites. In vitro maize seed infection assay with A. flavus showed up to 75% reduction of fungal growth in the infected RNAi seeds as compared to the seeds from control. A significant reduction was observed of aflatoxins (up to 86%) as well as another important fungal secondary metabolite called cyclopiazonic acid (CPA) (33-82%). f) Regeneration of transgenic cotton lines expressing selected resistant genes, for example, spot11 catalase, identified through comparative transcriptomic analysis (see Objective 2) is underway. g) Transgenic cotton lines expressing a synthetic peptide named D4E1 demonstrated antifungal effects against Aspergillus flavus under greenhouse conditions or seedling pathogens under field conditions. To ascertain the mode of action of the D4E1, experiments were conducted in collaboration with University of Louisiana, Lafayette, Louisiana, using a specialized dye (Sytox Green) to track movement of peptide molecules in fungal cells. ARS researchers demonstrated that the antimicrobial activity of D4E1 is due to membrane permeabilization and accumulation of reactive oxygen species (ROS) that induce cell death. In addition to developing crops resistant to aflatoxin contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral imaging technique (collecting and processing information from across the light-spectrum) to detect and quantify aflatoxins in corn kernels under Objective 4. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic Aspergillus flavus strains. A spectral signature to detect aflatoxin- contaminated corn has been developed and licensed. A prototype dual- camera based multispectral imaging system for rapid detection has been designed, developed, and evaluated for screening and removal technologies for automated grain handling systems in the U.S. and international markets. With funding from Gates Foundation and United States Agency for International Development (USAID) ARS researchers and collaborators developed a low cost portable technology to detect aflatoxin contamination in corn kernels for use in the developing countries. With additional funding from LaunchTN in Nashville, Tennessee, ARS researchers are in the process of assessing and refining a large version of an inspection device based on our �AflaGoggles" concept for screening aflatoxin contamination in maize - a quick and simple tool for detecting aflatoxin in corn especially in African countries. Funding from the National Science Foundation has also enabled a joint effort among Mississippi State University at Mississippi State, Mississippi, Secure Food Solutions, Inc. in Martin, Tennessee, and ARS researchers on a novel method to improve aflatoxin detection utilizing accuracy of multispectral imaging technology to develop commercial, rapid, screening equipment for aflatoxin-contaminated corn. Accomplishments 01 Host-induced gene silencing of fungal alpha-amylase to reduce infection and toxin production. Preharvest contamination of corn kernels with dangerous levels of aflatoxin not only reduces the value of the crop but also poses a health hazard to humans and livestock. Transgenic maize plants were generated by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, with an RNAi (ribonucleic acid interference) construct to silence the fungal alpha-amylase gene, which is essential for its growth and infection. As a result, fungal growth and aflatoxin production were significantly reduced (up to 98%). This technology, made possible by the short-lived RNA, does not require expression of a foreign protein so food produced from resistant lines of transgenic maize should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will serve as an excellent parent material to transfer the resistant trait to other commercial varieties. 02 Transfer of a natural antifungal protein from another plant to maize for controlling aflatoxin contamination. Preharvest contamination of corn kernels with dangerous levels of aflatoxin not only reduces the value of the crop but also poses a health hazard to humans and livestock. It has been established before that fungal alpha-amylase is essential for its growth and infection. Transgenic maize plants were regenerated expressing an antifungal protein from hyacinth beans that inhibits alpha-amylase in Aspergillus flavus. This research was conducted by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, in collaboration with University of Arkansas, Fayetteville, Arkansas. Kernels from sixth generation transgenic maize plants and their progenies were screened for their ability to withstand fungal infection and toxin production. Significant reduction was obtained in fungal growth and aflatoxin production (63-88%). Corn plants carrying this gene will serve as an excellent parent material to transfer the resistant trait to other commercial varieties. 03 Detection of aflatoxin contamination using a dual-camera based multispectral imaging system. Current screening methods for aflatoxin contamination in food and feed products is wrought with detection and sampling problems and the analysis by chemical methods is a time- consuming process. A prototype dual-camera based multispectral imaging system for the purpose of rapidly screening aflatoxin contamination has been designed, developed, and evaluated. This was achieved by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana. This technology enables rapid and non-destructive spectral- based aflatoxin contamination detection and removal in corn to provide toxin-free food supply.

Impacts
(N/A)

Publications

Mehanathan, M., Bedre, R., Mangu, V., Rajasekaran, K., Bhatnagar, D., Baisakh, N. 2018. Identification of candidate resistance genes of cotton against Aspergillus flavus infection using a comparative transcriptomics approach. Physiology and Molecular Biology of Plants. 24(3):513�519.
Sharma, K.K., Pothana, A., Prasad, K., Shah, D., Kaur, J., Bhatnagar, D., Chen, Z.-Y., Raruang, Y., Cary, J.W., Rajasekaran, K., Sudini, H.K., Bhatnagar-Mathur, P. 2017. Peanuts that keep aflatoxin at bay: a threshold that matters. Plant Biotechnology Journal. 16:1024-1033.
Majumdar, R., Rajasekaran, K., Sickler, C.M., Lebar, M.D., Musungu, B.M., Fakhoury, A.M., Payne, G.A., Geisler, M., Carter-Wientjes, C.H., Wei, Q., Bhatnagar, D., Cary, J.W. 2017. The pathogenesis-related maize seed (PRms) gene plays a role in resistance to Aspergillus flavus infection and aflatoxin contamination. Frontiers in Plant Science. 8:1758.
Tao, F., Yao, H., Hruska, Z., Burger, L.W., Rajasekaran, K., Bhatnagar, D. 2018. Recent development of optical methods in rapid and non-destructive detection of aflatoxin and fungal contamination in agricultural products. Trends in Analytical Chemistry. 100:65-81.
Rajasekaran, K., Sayler, R.J., Sickler, C.M., Majumdar, R., Jaynes, J.M., Cary, J.W. 2018. Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182. Plant Science. 270:150-156.
Bhatnagar, D., Rajasekaran, K., Gilbert, M.K., Cary, J.W., Magan, N. 2018. Advances in molecular and genomic research to safeguard food and feed supply from aflatoxin contamination. World Mycotoxin Journal. 11(1):47-72.
Gilbert, M.K., Majumdar, R., Rajasekaran, K., Chen, Z.-Y., Wei, Q., Sickler, C.M., Lebar, M.D., Cary, J.W., Frame, B.R., Wang, K. 2018. RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels. Planta. 247:1465�1473.
Moore, J., Rajasekaran, K., Cary, J.W., Chlan, C. 2017. Identification of resistance to Aspergillus flavus infection in cotton germplasm. Journal of Crop Improvement. 31(5):727-741.
Majumdar, R., Lebar, M.D., Mack, B.M., Minocha, R., Minocha, S., Carter- Wientjes, C.H., Sickler, C.M., Rajasekaran, K., Cary, J.W. 2018. The Aspergillus flavus spermidine synthase (spds) gene, is required for normal development, aflatoxin production, and pathogenesis during infection of maize kernels. Frontiers in Plant Science. 9:317.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance- associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus- host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms. Approach (from AD-416): Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over- expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally. Progress was made on all four objectives. The primary objective of this project is to identify native and foreign proteins or peptides (very small proteins) that provide resistance to the host crop (primarily corn and cotton) against fungal invasion and/or aflatoxin (compounds that are toxic and carcinogenic to humans and animals) contamination, and to test the efficacy of these genes by classical or molecular breeding of cotton and corn. Under Objective 1, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, made significant progress in identifying several native proteins in corn kernels that resist infection by the fungus, Aspergillus (A.) flavus that produces aflatoxins. Such resistance associated proteins and their corresponding genes have been identified in corn lines and these genes have been transferred to commercial varieties by classical breeding. After several field tests in the U.S. these varieties demonstrated aflatoxin-resistance comparable to known resistant lines. In collaboration with the International Institute of Tropical Agriculture, Nigeria, six corn lines (TZAR 101-106) were developed and they showed resistance not only to aflatoxin-producing fungi but also to a Fusarium fungus that produces another toxin called fumonisin. Using these resistant lines, several drought-tolerant lines were developed and they are being field/lab-tested in Nigeria/Southern Regional Research Center (SRRC). Under Objective 2, ARS scientists in New Orleans, Louisiana, have initiated experiments on a genome-wide transcriptome (the sum of all the actively expressed genes of a corn plant) analysis of the corn-A. flavus interaction. In collaboration with The J. Craig Venter Institute (JCVI) we will be using the modern ribonucleic acid-sequencing (RNA-Seq; a means of determining levels of activity of individual genes in both the fungus and corn) technique to study expression of genes during the corn-A. flavus interaction. We will be comparing A. flavus-infected kernels of TZAR 102 (a hybrid corn line derived from aflatoxin resistant germplasm developed in the U.S. and an African line that exhibits superior resistance to drought and ear rot), released by ARS-SRRC, along with a resistant line MI 82 and a susceptible check Va35 to delineate the molecular genetic differences that might explain the enhanced resistance to A. flavus observed in the TZAR 102 line. Corn seed has been infected with the A. flavus strain and we have collected the infected seed at 4 different time points (0, 8, 72 and 168 hours). The infected seed has been processed to stabilize the RNA needed for RNA-Seq analysis and the samples have been shipped to JCVI for sequencing. Data generated from RNA sequencing will be analyzed in-house. An interactome (the whole set of molecular interactions in a particular cell) analysis based on the RNA- Seq data will be performed enabling identification of key global regulators of A. flavus growth and aflatoxin biosynthesis as well as developmental and virulence (ability to cause infection) factors that can serve as targets for intervention strategies. This analysis will shed light specifically on the mechanisms of fungal pathogenesis and corn resistance. In cotton, we identified twenty-eight genes from infected cotton boll pericarp (outer coat) and seeds whose expression differed significantly compared to non-infected bolls. The Spot11 catalase gene showed up-regulation in inoculated locules (a segment of a cotton boll) and it is currently being evaluated in transgenic cotton. Recently, following a genome-wide transcriptome profiling that identified differentially expressed cotton genes in response to infection with both toxigenic and atoxigenic strains of A. flavus, a comparative transcriptome analysis was also performed that identified genes that were significantly differentially expressed in corn and peanut in response to A. flavus. We identified 732 unique genes with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes which could be used to improve resistance to aflatoxin resistance. Under Objective 3, we made significant progress in genetic engineering of corn and cotton to introduce genes for resistance to A. flavus growth and aflatoxin production. (a) Transgenic corn kernels expressing a synthetic peptide gene AGM 182 (modeled after an antimicrobial peptide from horseshoe crab) demonstrated a significant reduction in fungal growth and aflatoxin contamination (76- 98% reduction in third generation kernels). In a similar experiment, reduction in fungal growth and toxin production was also observed from sixth generation corn kernels expressing a gene from another plant, hyacinth beans, that encodes a novel antifungal seed protein. This antifungal protein inhibits a key enzyme (a-amylase) that is necessary for the fungus to grow and infect seed. (b) Significant progress has also been achieved in experiments to understand the contribution of native corn kernel proteins to A. flavus resistance. One such protein is called Pathogenesis�Related maize seed protein (PRms). To understand its role in fungal resistance the gene was first silenced using a ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) approach in transgenic corn lines. Down-regulation of PRms in transgenic kernels resulted in a ~250-350% increase in A. flavus growth accompanied by a 4.5 to 7.5-fold higher accumulation of aflatoxins than the control plants, confirming its central role in fungal resistance. This particular gene also affected the functioning of other genes believed to be involved in disease resistance. Similar RNAi-based approaches were carried out to silence fungal genes as well that are critical for the fungus to grow, infect and produce toxins. Our efforts to silence fungal genes such as fungal a-amylase (essential for fungal growth), VeA and NsdC [required by A. flavus for the production of aflatoxin and sclerotia (fungal survival structures)] have yielded aflatoxin resistance in transgenic kernels. (c) Unlike in corn, no resistant germplasm (seed stock) to A. flavus has been identified in cotton so far. ARS scientists in New Orleans, Louisiana, in collaboration with scientists at the University of Louisiana-Lafayette studied a wide variety of cotton germplasm to try and identify lines that demonstrate natural resistance to A. flavus infection and aflatoxin accumulation. Representative lines from three different species: Gossypium (G.) arboreum (old world cotton), G. barbadense (Pima cotton), and G. hirsutum (upland cotton) were screened with a green fluorescent protein (GFP) expressing A. flavus strain to assess any innate resistance to the fungus. Some cotton varieties of G. arboreum were the most resistant and commercially cultivated upland cotton varieties were moderately susceptible to A. flavus infection. (d) Substantial progress was also made on the correlation between cottonseed with rich reserves of lipids (a type of fat molecule that can serve as a nutrient source for fungi) and A. flavus infection and aflatoxin accumulation. Lipid accumulation in developing cottonseed was well-correlated with the ability of an aflatoxin-producing fungal strain to grow and produce aflatoxins compared to no toxin production by a non- toxin producing fungal strain. This study is useful to understand factors controlling A. flavus infection and aflatoxin production in developing cottonseed. (e) Transgenic cotton lines expressing a synthetic peptide designated D4E1 demonstrated antifungal effects against A. flavus under greenhouse conditions or seedling pathogens under field conditions. They were field- tested in collaboration with University of Arizona for resistance to aflatoxin contamination. Recent results from the small field experiment were inconclusive due to lack of A. flavus infection and aflatoxin contamination. Toxin detection procedures in corn and other crops are time consuming, destructive, inconsistent, and costly. Therefore, under Objective 4, ARS scientists in New Orleans, Louisiana, in collaboration with Geosystems Research Institute (GRI) of Mississippi State University (MSU) based at the Stennis Space Center, Mississippi, developed a non-invasive hyperspectral imaging technique (collecting and processing information from across the light-spectrum) to detect and quantify aflatoxins in corn kernels. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic A. flavus strains. A spectral signature to detect aflatoxin contaminated corn has been developed (U.S. Patent #8,563,934). A prototype multi-spectral imaging system for use in the field, as well as at grain inspection facilities has been designed. The patent has been licensed to Secure Food Solutions, Inc. (SFS), a Tennesse-based company, towards developing aflatoxin screening and removal technologies for automated grain handling systems in the U.S. and international markets. For this purpose, a Cooperative Research and Development Agreement was established on �Specialized high-resolution imaging system for rapid batch screening of aflatoxin in corn.� With funding from Gates Foundation and United States Agency for International Development we developed a portable technology to detect aflatoxin contamination in single corn cobs for farmers in the developing countries. We are also in the process of developing �AflaGoggles" for screening aflatoxin contamination in maize�-a quick and simple tool for detecting aflatoxin in corn especially in African countries. Funding from the National Science Foundation has also enabled a joint effort among SFS, MSU, and USDA on �Novel method to improve aflatoxin detection accuracy of multispectral imaging technology� to develop commercial, rapid, screening equipment for aflatoxin contaminated corn. Accomplishments 01 Development of aflatoxin and fumonisin-tolerant corn lines. Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, in collaboration with the International Institute of Tropical Agriculture, Nigeria developed six corn varieties (TZAR101-106) with resistance to contamination by the aflatoxin (compounds that are toxic and carcinogenic to humans and animals) producing fungus, Aspergillus flavus. In a recent field trial these six lines also demonstrated resistance to another fungus, Fusarium, responsible for producing a toxin called fumonisin. Using these lines several drought tolerant lines were also developed that will be used by growers in African countries to reduce the incidence of aflatoxin and fumonisin contamination in corn. 02 Aflatoxin-resistance conferred by the expression of a synthetic peptide (very small protein). Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, in collaboration with scientists at Tuskegee University and University of Arkansas developed transgenic corn lines expressing a synthetic antifungal peptide AGM 182 (modeled after an antimicrobial peptide from horseshoe crab). Transgenic corn kernels expressing AGM 182 demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). Transgenic corn lines with resistance to aflatoxin contamination will serve as a valuable germplasm for breeding new hybrids. 03 Demonstration of the role of a native corn kernel protein in aflatoxin resistance. The contribution of a native corn kernel protein, Pathogenesis�Related maize seed protein (PRms) to Aspergillus (A.) flavus resistance was validated by Agricultural Research Service (ARS) scientists in New Orleans, Louisiana. Using ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) -mediated silencing, the PRms gene was made ineffective in transgenic corn lines and the kernels were evaluated for A. flavus growth and aflatoxin contamination. Down- regulation of PRms in transgenic kernels resulted in a 250-350% increase in A. flavus growth accompanied by a 4.5 to 7.5-fold higher accumulation of aflatoxins than the control plants, confirming its central role in fungal resistance. This particular gene also affected the functioning of other genes believed to be involved in disease resistance in corn. Increased expression of PRms or breeding for it will provide resistance to aflatoxin contamination in transgenic kernels.

Impacts
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Publications

Rajasekaran, K., Ford, G.M., Sethumadhavan, K., Carter-Wientjes, C.H., Bland, J.M., Cao, H., Bhatnagar, D. 2017. Aspergillus flavus growth and aflatoxin production as influenced by total lipid content during growth and development of cottonseed. Journal of Crop Improvement. 31(1):91-99.
Majumdar, R., Rajasekaran, K., Cary, J.W. 2017. RNA interference (RNAi) as a potential tool for control of mycotoxin contamination in crop plants: concepts and considerations. Frontiers in Plant Science. 8:200. doi:10. 3389/fpls.2017.00200.
Anderson, D.M., Rajasekaran, K. 2016. The global importance of transgenic cotton. In: Ramawat, K.G., Ahuja, M.R., editors. Fiber Plants: Biology, Biotechnology and Applications, Volume 13. Switzerland: Springer International Publishing. p. 17-33. doi:10.1007/978-3-319-44570-0_2.
Brown, R.L., Williams, W.P., Windham, G.L., Menkir, A., Chen, Z.-Y. 2016. Evaluation of African-bred maize germplasm lines for resistance to aflatoxin accumulation. Agronomy. 6(2):24. doi:10.3390/agronomy6020024.
Srour, A.Y., Fakhoury, A.M., Brown, R.L. 2016. Targeting mycotoxin biosynthesis pathway genes. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi: Methods and Protocols, Methods in Molecular Biology. New York, NY: Springer. 1542:159-171.
Sakhanokho, H.F., Rajasekaran, K. 2016. Cotton regeneration in vitro. In: Ramawat, K.G., Ahuja, M.R., editors. Fiber Plants: Biology, Biotechnology and Applications. Gewerbestrasse, Switzerland: Springer International Publishing AG. p. 87-110.
Rajasekaran, K., Majumdar, R., Sickler, C.M., Wei, Q., Cary, J.W., Bhatnagar, D. 2017. Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene, bar. Journal of Crop Improvement. 31(4):628-636. doi:10.1080/15427528.2017. 1327913.