Responses of wheat (Triticum aestivum L.) constitutively expressing four different monolignol biosynthetic genes to Fusarium head blight caused by Fusarium graminearum.

The Fusarium head blight (FHB) pathogen Fusarium graminearum produces the trichothecene mycotoxin deoxynivalenol (DON) and reduces wheat yield and grain quality. Spring wheat (Triticum aestivum L.) genotype CB037 was transformed with constitutive expression (CE) constructs containing sorghum (Sorghum bicolor L. (Moench)) genes encoding monolignol biosynthetic enzymes, caffeoyl-Coenzyme A (CoA) 3-O-methyltransferase (SbCCoAOMT), 4-coumarate-CoA ligase (Sb4CL), or coumaroyl shikimate 3-hydroxylase (SbC3'H), or monolignol pathway transcriptional activator, SbMyb60. Spring wheats were screened for Type I (resistance to initial infection, using spray inoculations) and Type II (resistance to spread within the spike, using single floret inoculations) resistances in the field (spray) and greenhouse (spray and single floret). Following field inoculations, disease index, percent Fusarium damaged kernels (FDK), and DON measurements of CE plants were similar to or greater than CB037. For greenhouse inoculations, the area under the disease progress curve (AUDPC) and FDK were determined. Following screens, focus was placed on two each, SbC3'H and SbCCoAOMT CE lines because of trends towards decreased AUDPC and FDK observed following single floret inoculations. These four lines were as susceptible as CB037 following spray inoculations. However, single floret inoculations showed that these CE lines had significantly reduced AUDPC (P<0.01) and FDK (P≤0.02) compared with CB037, indicating improved Type II resistance. None of these CE lines had increased acid detergent lignin, as compared with CB037, indicating that lignin concentration may not be a major factor in FHB resistance. The SbC3'H and SbCCoAOMT CE lines are valuable for investigating phenylpropanoid-based resistance to FHB.

Phenylpropanoids Following Wounding and Infection of Sweet Sorghum Lines Differing in Responses to Stalk Pathogens.

Sweet sorghum (Sorghum bicolor) lines M81-E and Colman were previously shown to differ in responses to Fusarium thapsinum and Macrophomina phaseolina, stalk rot pathogens that can reduce the yields and quality of biomass and extracted sugars. Inoculated tissues were compared for transcriptomic, phenolic metabolite, and enzymatic activity during disease development 3 and 13 days after inoculation (DAI). At 13 DAI, M81-E had shorter mean lesion lengths than Colman when inoculated with either pathogen. Transcripts encoding monolignol biosynthetic and modification enzymes were associated with transcriptional wound (control) responses of both lines at 3 DAI. Monolignol biosynthetic genes were differentially coexpressed with transcriptional activator SbMyb76 in all Colman inoculations, but only following M. phaseolina inoculation in M81-E, suggesting that SbMyb76 is associated with lignin biosynthesis during pathogen responses. In control inoculations, defense-related genes were expressed at higher levels in M81-E than Colman. Line, treatment, and timepoint differences observed in phenolic metabolite and enzyme activities did not account for observed differences in lesions. However, generalized additive models were able to relate metabolites, but not enzyme activities, to lesion length for quantitatively modeling disease progression: in M81-E, but not Colman, sinapic acid levels positively predicted lesion length at 3 DAI when cell wall-bound syringic acid was low, soluble caffeic acid was high, and lactic acid was high, suggesting that sinapic acid may contribute to responses at 3 DAI. These results provide potential gene targets for development of sweet sorghum varieties with increased stalk rot resistance to ensure biomass and sugar quality.

Effects of Altering Three Steps of Monolignol Biosynthesis on Sorghum Responses to Stalk Pathogens and Water Deficit.

The drought-resilient crop sorghum (Sorghum bicolor [L.] Moench) is grown worldwide for multiple uses, including forage or potential lignocellulosic bioenergy feedstock. A major impediment to biomass yield and quality are the pathogens Fusarium thapsinum and Macrophomina phaseolina, which cause Fusarium stalk rot and charcoal rot, respectively. These fungi are more virulent with abiotic stresses such as drought. Monolignol biosynthesis plays a critical role in plant defense. The genes Brown midrib (Bmr)6, Bmr12, and Bmr2 encode the monolignol biosynthesis enzymes cinnamyl alcohol dehydrogenase, caffeic acid O-methyltransferase, and 4-coumarate:CoA ligase, respectively. Plant stalks from lines overexpressing these genes and containing bmr mutations were screened for pathogen responses with controlled adequate or deficit watering. Additionally, near-isogenic bmr12 and wild-type lines in five backgrounds were screened for response to F. thapsinum with adequate and deficit watering. All mutant and overexpression lines were no more susceptible than corresponding wild-type under both watering conditions. The bmr2 and bmr12 lines, near-isogenic to wild-type, had significantly shorter mean lesion lengths (were more resistant) than RTx430 wild-type when inoculated with F. thapsinum under water deficit. Additionally, bmr2 plants grown under water deficit had significantly smaller mean lesions when inoculated with M. phaseolina than under adequate-water conditions. When well-watered, bmr12 in cultivar Wheatland and one of two Bmr2 overexpression lines in RTx430 had shorter mean lesion lengths than corresponding wild-type lines. This research demonstrates that modifying monolignol biosynthesis for increased usability may not impair plant defenses but can even enhance resistance to stalk pathogens under drought conditions.

Pathogen and drought stress affect cell wall and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant.

BACKGROUND: As effects of global climate change intensify, the interaction of biotic and abiotic stresses increasingly threatens current agricultural practices. The secondary cell wall is a vanguard of resistance to these stresses. Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot) cause internal damage to the stalks of the drought tolerant C4 grass, sorghum (Sorghum bicolor (L.) Moench), resulting in reduced transpiration, reduced photosynthesis, and increased lodging, severely reducing yields. Drought can magnify these losses. Two null alleles in monolignol biosynthesis of sorghum (brown midrib 6-ref, bmr6-ref; cinnamyl alcohol dehydrogenase, CAD; and bmr12-ref; caffeic acid O-methyltransferase, COMT) were used to investigate the interaction of water limitation with F. thapsinum or M. phaseolina infection. RESULTS: The bmr12 plants inoculated with either of these pathogens had increased levels of salicylic acid (SA) and jasmonic acid (JA) across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering, which suggested that drought may prime induction of pathogen resistance. RNA-Seq analysis revealed coexpressed genes associated with pathogen infection. The defense response included phytohormone signal transduction pathways, primary and secondary cell wall biosynthetic genes, and genes encoding components of the spliceosome and proteasome. CONCLUSION: Alterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in SA and JA signal transduction may be involved in priming a stronger defense response in water-limited bmr12 plants.

Response of Sorghum Enhanced in Monolignol Biosynthesis to Stalk Rot Pathogens.

To increase phenylpropanoid constituents and energy content in the versatile C4 grass sorghum (Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3-O-methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum-inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina-inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum. Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60, Bmr2, or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Amylose-Free ("waxy") Wheat Colonization by Fusarium spp. and Response to Fusarium Head Blight.

Hexaploid waxy wheat (Triticum aestivum L.) has null mutations in Wx genes and grain lacking amylose with increased digestibility and usability for specialty foods. The waxy cultivar Mattern is susceptible to Fusarium head blight (FHB) caused by Fusarium graminearum species complex, which produces the mycotoxin deoxynivalenol (DON). In experiment 1, conducted during low natural FHB, grain from waxy breeding lines, Mattern, and wild-type breeding lines and cultivars were assessed for Fusarium infection and DON concentration. Nine Fusarium species and species complexes were detected from internally infected (disinfested) grain; F. graminearum infections were not different between waxy and wild-type. Surface- and internally infected grain (nondisinfested) had greater numbers of Fusarium isolates across waxy versus wild-type, but F. graminearum-like infections were similar; however, DON levels were higher in waxy. In experiment 2, conducted during a timely epidemic, disease severity, Fusarium-damaged kernels (FDK), and DON were assessed for waxy breeding lines, Mattern, and wild-type cultivars. Disease severity and FDK were not significantly different from wild-type, but DON was higher in waxy than wild-type lines. Across both experiments, waxy breeding lines, Plant Introductions 677876 and 677877, responded similarly to FHB as moderately resistant wild-type cultivar Overland, showing promise for breeding advanced waxy cultivars with reduced FHB susceptibility.

Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (FST) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. FST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

Response of Sweet Sorghum Lines to Stalk Pathogens Fusarium thapsinum and Macrophomina phaseolina.

Sweet sorghum (Sorghum bicolor (L.) Moench) has potential for bioenergy. It is adapted to a variety of U.S. locations and the extracted juice can be directly fermented into ethanol. However, little research on fungal stalk rots, diseases that pose serious constraints for yield and quality of juice and biomass, has been reported. A greenhouse bioassay was designed to assess charcoal rot (Macrophomina phaseolina) and Fusarium stalk rot (Fusarium thapsinum) in plants at maturity, the developmental stage at which these diseases are manifested. Multiple plantings of a susceptible grain line, RTx430, were used as a control for variation in flowering times among sweet sorghum lines. Lesion length measurements in inoculated peduncles were used to quantify disease severity. Sweet sorghum lines 'Rio' and 'M81E' exhibited resistance to F. thapsinum and M. phaseolina, respectively; and, in contrast, 'Colman' sorghum exhibited susceptibility to both pathogens. Lesion development over time in Colman was monitored. These results will enhance molecular and biochemical analyses of responses to pathogens, and breeding stalk-rot-resistant sweet sorghum lines.

Effect of waxy (Low Amylose) on Fungal Infection of Sorghum Grain.

Loss of function mutations in waxy, encoding granule bound starch synthase (GBSS) that synthesizes amylose, results in starch granules containing mostly amylopectin. Low amylose grain with altered starch properties has increased usability for feed, food, and grain-based ethanol. In sorghum, two classes of waxy (wx) alleles had been characterized for absence or presence of GBSS: wx(a) (GBSS(-)) and wx(b) (GBSS(+), with reduced activity). Field-grown grain of wild-type; waxy, GBSS(-); and waxy, GBSS(+) plant introduction accessions were screened for fungal infection. Overall, results showed that waxy grains were not more susceptible than wild-type. GBSS(-) and wild-type grain had similar infection levels. However, height was a factor with waxy, GBSS(+) lines: short accessions (wx(b) allele) were more susceptible than tall accessions (undescribed allele). In greenhouse experiments, grain from accessions and near-isogenic wx(a), wx(b), and wild-type lines were inoculated with Alternaria sp., Fusarium thapsinum, and Curvularia sorghina to analyze germination and seedling fitness. As a group, waxy lines were not more susceptible to these pathogens than wild-type, supporting field evaluations. After C. sorghina and F. thapsinum inoculations most waxy and wild-type lines had reduced emergence, survival, and seedling weights. These results are valuable for developing waxy hybrids with resistance to grain-infecting fungi.