Occurrence in Seeds and Potential Seed Transmission of Xanthomonas vasicola pv. vasculorum in Maize in the United States.

This paper reports original evidence regarding the potential role of seed transmission of Xanthomonas vasicola pv. vasculorum in the epidemiology of bacterial leaf streak (BLS) in maize. We evaluated the occurrence of the pathogen on seeds from diseased fields and its subsequent transmission to seedlings. In 2016 and 2017, X. vasicola pv. vasculorum was detected by TaqMan PCR from 22 of 41 maize seed lots harvested from naturally infected fields in Colorado, Nebraska, and Iowa. However, many of the PCR-positive samples did not yield culturable X. vasicola pv. vasculorum colonies. The highest levels of seed contamination were detected in dent maize and popcorn from NE and CO. Seed transmission was evaluated in greenhouse grow-outs from eight seed lots, totaling more than 14,000 plants. Putative seed transmission events from naturally contaminated seed lots, estimated from PCR results, occurred at a frequency between 0.1 and 0.5% in 10-seedling pooled samples and at a frequency of 2.7% from individual plant assays. However, no seedling symptoms were observed during these assays and live X. vasicola pv. vasculorum colonies were not recovered from PCR-positive seedlings. In contrast, seed transmission was readily demonstrated from artificially contaminated seed lots, including typical symptoms and recovery of live bacteria. Seed transmission consistently occurred from seeds soaked in bacterial suspensions with concentrations of ≥106 CFU/ml, suggesting that a threshold population of the bacterium is necessary for the development of BLS symptoms and recovery of live bacteria. The low bacterial populations on naturally contaminated seeds apparently were not sufficient to result in diseased seedlings.

Effects of Temperature and pH on Fusarium oxysporum and Soybean Seedling Disease.

Fusarium oxysporum (Fo) is an important pathogen that reduces soybean yield by causing seedling disease and root rot. This study assessed the effects of pH and temperature on Fo fungal growth and seedling disease. In an in vitro assay, 14 Fo isolates collected from symptomatic soybean roots across Iowa in 2007 were grown on artificial culture media at five pH levels (4, 5, 6, 7, and 8) and incubated at four temperatures (15, 20, 25, or 30°C). In a rolled-towel assay, soybean seeds from Fo-susceptible cultivar Jack were inoculated with a suspension of a pathogenic or a nonpathogenic Fo isolate; both isolates were previously designated for their relative aggressiveness in causing root rot at 25°C. The seeds were placed in rolled germination paper, and the rolls were incubated in all combinations of buffer solutions at four pH levels (4, 5, 6, and 7), and four temperatures (15, 20, 25, or 30°C). There was a significant interaction between temperature and pH (P < 0.05) for in vitro radial growth and root rot severity. Isolates showed the most in vitro radial growth after incubation at pH 6 and 25°C. For the rolled-towel assay, the pathogenic isolate caused the most severe root rot at pH 6 and 30°C. Gaussian regression analysis estimates for optimal conditions were pH 6.3 at 27.1°C for maximal fungal growth and pH 5.9 at 30°C for maximal root rot severity. These results indicate that optimal pH and temperature conditions are similar for Fo growth and disease in soybean seedlings and suggest that Fo may be a more important seedling pathogen when soybeans are planted under warm conditions in moderately acidic soils.

Roles of Genotype-Determined Mycotoxins in Maize Seedling Blight Caused by Fusarium graminearum.

Fusarium graminearum is an important causal agent of maize seedling blight. The species includes several chemotypes that produce various forms of deoxynivalenol (DON) and nivalenol (NIV). To understand the effects and roles of F. graminearum mycotoxins on maize seedling blight occurring at Zhang Ye of Gansu, China, 23 isolates of F. graminearum were collected and characterized. A PCR assay showed all 23 isolates belonged to the 15-acetyldeoxynivalenol (15-ADON) genotype. This was also confirmed by production of both DON and 15-ADON in either rice culture medium or maize seedling roots, detected by high performance liquid chromatography and mass spectrometry. In maize seedling roots, 15-ADON dominated at 6 days post inoculation (dpi) and DON was the main mycotoxin at 12 dpi. The biomass of F. graminearum doubled from 6 to 12 dpi, and was positively correlated with virulence of the isolates. Both mycotoxins affected maize root vitality, but 15-ADON had a greater effect than DON. ALDH9 and MDH, two dehydrogenase synthesis genes in maize, showed a lower relative expression in 15-ADON treatments than in DON treatments. It indicated that both mycotoxins affected seed germination and root development, with 15-ADON being more destructive. Under scanning electron microscopy and transmission electron microscopy, root hair formation and development were delayed by DON, but completely inhibited by 15-ADON. 15-ADON caused cell shrinkage, loose cellular structure, and widened intercellular spaces; it also destroyed organelles and caused plasmolysis, and eventually ruptured cell membranes causing cell death. DON did not affect cell morphology and arrangement, but altered the morphology of organelles, forming concentric membranous bodies and a large amount of irregular lipid droplets. Thus, both mycotoxins contributed to symptom expression of maize seedling blight, but 15-ADON was more destructive than DON.

Association of Putative Fungal Effectors in Fusarium oxysporum with Wilt Symptoms in Soybean.

Fungi within the Fusarium oxysporum species complex can cause root rot, seedling blight, and wilt of soybean. Isolates recovered from soybean vary in aggressiveness and also the type of symptoms they produce. The aim of this study was to identify genetic markers to detect aggressive soybean wilt isolates. Eighty isolates collected primarily from soybean were tested in the greenhouse for their ability to produce wilt symptoms using susceptible 'Jack' soybean. The same 80 isolates were assessed for the presence of fungal effector genes Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, and Snf1. All polymerase chain reaction amplicons were sequenced, phylogenies were inferred, and analysis of molecular variance (AMOVA) was performed for 10 of the 12 genes. High incidence of vascular discoloration of roots or stems was observed with 3 isolates, while moderate to low levels of incidence were observed for 25 isolates. Fungal effector genes Fmk1, Fow1, PelA, Rho1, Sge1, and Snf1 were present in all isolates screened, while Pda1, PelD, Pep1, Prt1, Six1, and Six6 were dispersed among isolates. The Bayesian and AMOVA analyses found that the genes Fmk1, Fow1, Pda1, PelA, Rho1, Sge1, and Snf1 corresponded to previously designated clades based on tef1α and mitochondrial small subunit sequences. None of the genes had a significant association with wilt symptoms on soybean. Interestingly, the Six6 gene was only present in three previously known wilt isolates from soybean, common bean, and tomato; of these, the soybean and common bean isolates produced high levels of vascular wilt in our study.

Association of Effector Six6 with Vascular Wilt Symptoms Caused by Fusarium oxysporum on Soybean.

The Fusarium oxysporum species complex (FOSC) is a widely distributed group of fungi that includes both pathogenic and nonpathogenic isolates. In a previous study, isolates within the FOSC collected primarily from soybean were assessed for the presence of 12 fungal effector genes. Although none of the assayed genes was significantly associated with wilt symptoms on soybean, the secreted in xylem 6 (Six6) gene was present only in three isolates, which all produced high levels of vascular wilt on soybean. In the current study, a collection of F. oxysporum isolates from soybean roots and F. oxysporum f. sp. phaseoli isolates from common bean was screened for the presence of the Six6 gene. Interestingly, all isolates for which the Six6 amplicon was generated caused wilt symptoms on soybean, and two-thirds of the isolates showed high levels of aggressiveness, indicating a positive association between the presence of the effector gene Six6 and induction of wilt symptoms. The expression profile of the Six6 gene analyzed by quantitative reverse-transcription polymerase chain reaction revealed an enhanced expression for the isolates that caused more severe wilt symptoms on soybean, as established by the greenhouse assay. These findings suggest the suitability of the Six6 gene as a possible locus for pathogenicity-based molecular diagnostics across the various formae speciales.

Transcriptome profiling of soybean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is one of the most common fungal pathogens causing soybean root rot and seedling blight in U.S.A. In a recent study, significant variation in aggressiveness was observed among isolates of F. oxysporum collected from roots in Iowa, ranging from highly pathogenic to weakly or non-pathogenic isolates. RESULTS: We used RNA-seq analysis to investigate the molecular aspects of the interactions of a partially resistant soybean genotype with non-pathogenic/pathogenic isolates of F. oxysporum at 72 and 96 h post inoculation (hpi). Markedly different gene expression profiles were observed in response to the two isolates. A peak of highly differentially expressed genes (HDEGs) was triggered at 72 hpi in soybean roots and the number of HDEGs was about eight times higher in response to the pathogenic isolate compared to the non-pathogenic one (1,659 vs. 203 HDEGs, respectively). Furthermore, the magnitude of induction was much greater in response to the pathogenic isolate. This response included a stronger activation of defense-related genes, transcription factors, and genes involved in ethylene biosynthesis, secondary and sugar metabolism. CONCLUSIONS: The obtained data provide an important insight into the transcriptional responses of soybean-F. oxysporum interactions and illustrate the more drastic changes in the host transcriptome in response to the pathogenic isolate. These results may be useful in the developing new methods of broadening resistance of soybean to F. oxysporum, including the over-expression of key soybean genes.

Genotypic and Phenotypic Characterization of Fungi in the Fusarium oxysporum Species Complex from Soybean Roots.

Isolates in the Fusarium oxysporum species complex (FOSC) from soybean range from nonpathogenic to aggressive pathogens causing seedling damping-off, wilt, and root rot. The objective of this research was to characterize the genotype and phenotype of isolates within the FOSC recovered predominantly from soybean roots and seedlings. Sequence analyses of the translation elongation factor (tef1α) gene and the mitochondrial small subunit (mtSSU), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the intergenic spacer (IGS) region, and identification of the mating type loci were conducted for 170 isolates. Vegetative compatibility (VC) tests were conducted for 114 isolates. Isolate aggressiveness was tested using a rolled towel assay for 159 isolates. Phylogenetic analysis of the tef1α and mtSSU and PCR-RFLP analysis of the IGS region separated the FOSC isolates into five clades, including F. commune. Both mating type loci, MAT1-1 or MAT1-2, were present in isolates from all clades. The VC tests were not informative, because most VC groups consisted of a single isolate. Isolate aggressiveness varied within and among clades; isolates in clade 2 were significantly less aggressive (P < 0.0001) when compared with isolates from the other clades and F. commune. The results from this study demonstrate the high levels of genotypic and phenotypic diversity within the FOSC from soybean but further work is needed to identify characteristics associated with pathogenic capabilities.

Aggressiveness of Fusarium species and impact of root infection on growth and yield of soybeans.

Fusarium spp. are commonly isolated from soybean roots but the pathogenic activity of most species is poorly documented. Aggressiveness and yield impact of nine species of Fusarium were determined on soybean in greenhouse (50 isolates) and field microplot (19 isolates) experiments. Root rot severity and shoot and root dry weights were compared at growth stages V3 or R1. Root systems were scanned and digital image analysis was conducted; yield was measured in microplots. Disease severity and root morphology impacts varied among and within species. Fusarium graminearum was highly aggressive (root rot severity >90%), followed by F. proliferatum and F. virguliforme. Significant variation in damping-off (20 to 75%) and root rot severity (<20 to >60%) was observed among F. oxysporum isolates. In artificially-infested microplots, root rot severity was low (<25%) and mean yield was not significantly reduced. However, there were significant linear relationships between yield and root symptoms for some isolates. Root morphological characteristics were more consistent indicators of yield loss than root rot severity. This study provides the first characterization of aggressiveness and yield impact of Fusarium root rot species on soybean at different plant stages and introduces root image analysis to assess the impact of root pathogens on soybean.

Seed pathology progress in academia and industry.

Seed pathology involves the study and management of diseases affecting seed production and utilization, as well as disease management practices applied to seeds. In this paper, three aspects of seed pathology are discussed: research innovations in detection of seedborne pathogens and elucidation of their epidemiology; advances in development and use of seed treatments; and progress toward standardization of phytosanitary regulations and seed health testing methods. The application of nucleic-acid based detection methods in seed health testing has been facilitated by integrating conventional or real-time PCR with other technologies (e.g., BIO-PCR, IMS-PCR, MCH-PCR). PCR-based methods and pathogen marker technologies are being applied to epidemiological research on seedborne pathogens, e.g., seed transmission mechanisms, the influence of external biotic and abiotic factors on seed transmission, and tracking progress of seed-transmitted pathogens. Seed treatment use is discussed in terms of the revolutionary expansion in seed-applied insecticide use, impacts of new fungicide active ingredients, and the effects of some seed treatments on crop physiology. International seed trade has been affected significantly by changing phytosanitary regulations, not always based on science. Efforts are underway to revise phytosanitary regulations to reflect pest risk analysis outcomes and to develop standards for seed health testing methods that facilitate safe and efficient international trade in seeds.

Mycotoxin Production in Fusarium According to Contemporary Species Concepts.

Fusarium is one of the most important genera of plant-pathogenic fungi in the world and arguably the world's most important mycotoxin-producing genus. Fusarium species produce a staggering array of toxic metabolites that contribute to plant disease and mycotoxicoses in humans and other animals. A thorough understanding of the mycotoxin potential of individual species is crucial for assessing the toxicological risks associated with Fusarium diseases. There are thousands of reports of mycotoxin production by various species, and there have been numerous attempts to summarize them. These efforts have been complicated by competing classification systems based on morphology, sexual compatibility, and phylogenetic relationships. The current depth of knowledge of Fusarium genomes and mycotoxin biosynthetic pathways provides insights into how mycotoxin production is distributedamong species and multispecies lineages (species complexes) in the genus as well as opportunities to clarify and predict mycotoxin risks connected with known and newly described species. Here, we summarize mycotoxin production in the genus Fusarium and how mycotoxin risk aligns with current phylogenetic species concepts.

Mycotoxins in ethanol co-products: modeling economic impacts on the livestock industry and management strategies.

The rapidly expanding U.S. ethanol industry is generating a growing supply of co-products, mostly in the form of dried distillers' grain and solubles (DDGS) or wet distillers' grains (WDG). In the United States, 90% of the co-products of maize-based ethanol are fed to livestock. An unintended consequence is that animals are likely to be fed higher levels of mycotoxins, which are concentrated up to three times in DDGS compared to grain. The model developed in this study estimates current losses to the swine industry from weight gain reduction due to fumonisins in added DDGS at $9 million ($2-18 million) annually. If there is complete market penetration of DDGS in swine feed with 20% DDGS inclusion in swine feed and fumonisins are not controlled, losses may increase to $147 million ($29-293 million) annually. These values represent only those losses attributable to one mycotoxin on one adverse outcome on one species. The total loss due to mycotoxins in DDGS could be significantly higher due to additive or multiplicative effects of multiple mycotoxins on animal health. If mycotoxin surveillance is implemented by ethanol producers, losses are shifted among multiple stakeholders. Solutions to this problem include methods to reduce mycotoxin contamination in both pre- and postharvest maize.

Reduced Incidence of Bacterial Rot on Transgenic Insect-Resistant Maize in the Philippines.

In the Philippines and parts of Southeast Asia, Asian corn borer (Ostrinia furnacalis) is a serious pest of maize, and injury from this insect often is associated with the occurrence of bacterial stalk and ear rot (caused by Erwinia chrysanthemi pv. zeae). The effect of transgenic insect protection on the incidence of bacterial stalk and ear rot was studied in the Philippines with seven field trials in Mindanao and two trials in Laguna. Three transgenic hybrids (expressing Bt protein Cry1Ab) and their conventional near-isogenic counterparts were included in Mindanao, and one transgenic/conventional hybrid pair was used in Laguna (Los Banos). In the Mindanao trials, bacterial stalk rot was rated on a 1 to 9 scale approximately 2 weeks before harvest, while in Laguna, bacterial rot mortality and bacterial ear rot incidence were assessed 10 days before and at harvest, respectively. In all trials, the number of Asian corn borer tunnels was assessed by splitting stalks at harvest. Results of the trials showed significant differences between the transgenic hybrids and their conventional counterparts in terms of bacterial stalk and ear rot incidence, number of Asian corn borer tunnels, and yield. Transgenic hybrids invariably showed significantly lower bacterial stalk rot mortality and ear rot incidence, no Asian corn borer infestation, and higher yield compared with their conventional counterparts. Average yield advantage of transgenic hybrids ranged from 1.2 to 5.1 t/ha. Results confirm the important role of Asian corn borer in the initiation and spread of bacterial stalk and ear rot in maize; hence, the use of transgenic insect-resistant hybrids will have an added value in areas where this disease is prevalent.

Fusarium Species and Their Associated Mycotoxins.

The genus Fusarium includes numerous toxigenic species that are pathogenic to plants or humans, and are able to colonize a wide range of environments on earth. The genus comprises around 70 well-known species, identified by using a polyphasic approach, and as many as 300 putative species, according to phylogenetic species concepts; many putative species do not yet have formal names. Fusarium is one of the most economically important fungal genera because of yield loss due to plant pathogenic activity; mycotoxin contamination of food and feed products which often render them unaccep for marketing; and health impacts to humans and livestock, due to consumption of mycotoxins. Among the most important mycotoxins produced by species of Fusarium are the trichothecenes and the fumonisins. Fumonisins cause fatal livestock diseases and are considered potentially carcinogenic mycotoxins for humans, while trichothecenes are potent inhibitors of protein synthesis. This chapter summarizes the main aspects of morphology, pathology, and toxigenicity of the main Fusarium species that colonize different agricultural crops and environments worldwide, and cause mycotoxin contamination of food and feed.

Cultural and genetic approaches to managing mycotoxins in maize.

Infection of maize kernels by toxigenic fungi remains a challenging problem despite decades of research progress. Cultural practices, including crop rotation, tillage, planting date, and management of irrigation and fertilization, have limited effects on infection and subsequent mycotoxin accumulation. Current infrastructure and grain storage practices in developed countries can prevent postharvest development of mycotoxins, but this aspect remains a threat in developing countries, especially in tropical areas. Because most mycotoxin problems develop in the field, strategies are needed to prevent infection of growing plants by toxigenic fungi. Developing genetic resistance to Aspergillus flavus, Gibberella zeae, and Fusarium spp. (particularly F. verticillioides) in maize is a high priority. Sources of resistance to each of these pathogens have been identified and have been incorporated into public and private breeding programs. However, few, if any, commercial cultivars have adequate levels of resistance. Efforts to control infection or mycotoxin development through conventional breeding and genetic engineering are reviewed. The role of transgenic insect control in the prevention of mycotoxins in maize is discussed.

Comparison of Fumonisin Concentrations in Kernels of Transgenic Bt Maize Hybrids and Nontransgenic Hybrids.

Maize hybrids genetically engineered with genes from the bacterium Bacillus thuringiensis (Bt maize) express CryIA(b) and other Cry proteins that are toxic to certain insects, particularly the European corn borer (Ostrinia nubilalis). Maize kernel feeding by O. nubilalis often leads to infection by fungi in the genus Fusarium, including the fumonisin-producing species F. verticillioides and F. proliferatum. In field experiments in 1995, 1996, and 1997, transgenic maize hybrids and near-isogenic, nontransgenic hybrids were manually infested with neonatal European corn borer larvae. Manual infestation increased Fusarium ear rot severity and fumonisin concentrations in kernels of nontransgenic hybrids. Transgenic hybrids with kernel expression of CryIA(b) consistently experienced less insect feeding on kernels and less Fusarium ear rot than their nontransgenic counterparts. In manually infested treatments, these hybrids also exhibited lower concentrations of fumonisins in kernels compared with their nontransgenic counterparts. In manually infested treatments in 1995, mean fumonisin B1 concentrations were 8.8 µg/g in the nontransgenic hybrid and 6.7 or 3.0 µg/g in transgenic hybrids. In 1996, mean fumonisin B1 concentrations in manually infested treatments were 4.9 µg/g (range 2.3 to 8.8) for nontransgenic and 1.2 µg/g (range 1.0 to 1.3) for transgenic hybrids with kernel expression. Mean total fumonisin concentrations (fumonisin B1 + B2 + B3) were 7.0 µg/g (range 3.0 to 12.2) for nontransgenic and 1.7 µg/g (range 1.5 to 1.9) for transgenic hybrids with kernel expression. In 1997, mean fumonisin B1 concentrations in manually infested treatments were 11.8 µg/g (range 7.6 to 17.3) for nontransgenic and 1.3 µg/g (range 0.8 to 2.2) for transgenic hybrids with kernel expression of CryIA(b) or Cry9C. Mean total fumonisin concentrations were 16.5 µg/g (range 10.7 to 24.0) for nontransgenic and 2.1 µg/g (range 1.5 to 3.1) for transgenic hybrids with kernel expression. Transgenic hybrids that do not express CryIA(b) or Cry9C in kernels did not consistently have fumonisin concentrations different from the nontransgenic hybrids. Higher fumonisin concentrations in nontransgenic hybrids were associated with high European corn borer populations during the early reproductive stages of the maize plants. These results indicate that under some conditions, genetic engineering of maize for insect resistance may enhance its safety for animal and human consumption.

Fumonisins in conventional and transgenic, insect-resistant maize intended for fuel ethanol production: implications for fermentation efficiency and DDGS co-product quality.

Mycotoxins in maize grain intended for ethanol production are enriched in co-product dried distiller's grains and solubles (DDGS) and may be detrimental to yeast in fermentation. This study was conducted to examine the magnitude of fumonisin enrichment in DDGS and to analyze the impacts of insect injury, Fusarium ear rot severity, and fumonisin contamination on final ethanol yield. Samples of naturally-contaminated grain (0 to 35 mg/kg fumonisins) from field trials conducted in 2008-2011 were fermented and DDGS collected and analyzed for fumonisin content. Ethanol yield (determined gravimetrically) was unaffected by fumonisins in the range occurring in this study, and was not correlated with insect injury or Fusarium ear rot severity. Ethanol production was unaffected in fumonisin B1-spiked grain with concentrations from 0 to 37 mg/kg. Bacillus thuringiensis (Bt) maize often has reduced fumonisins due to its protection from insect injury and subsequent fungal infection. DDGS derived from Bt and non-Bt maize averaged 2.04 mg/kg and 8.25 mg/kg fumonisins, respectively. Fumonisins were enriched by 3.0× for 50 out of 57 hybrid × insect infestation treatment combinations; those seven that differed were <3.0 (1.56 to 2.56×). This study supports the industry assumption of three-fold fumonisin enrichment in DDGS, with measurements traceable to individual samples. Under significant insect pest pressures, DDGS derived from Bt maize hybrids were consistently lower in fumonisins than DDGS derived from non-Bt hybrids.