Quantitative trait loci for resistance to black point caused by Bipolaris sorokiniana in bread wheat.

Black point disease is a serious concern in wheat production worldwide. In this study, we aimed to identify the major quantitative trait loci (QTL) for resistance to black point caused by Bipolaris sorokiniana and develop molecular markers for marker-assisted selection (MAS). A recombinant inbred line (RIL) population derived from a cross between PZSCL6 (highly susceptible) and Yuyou1 (moderately resistant) was evaluated for black point resistance at four locations under artificial inoculation with B. sorokiniana. Thirty resistant and 30 susceptible RILs were selected to form resistant and susceptible bulks, respectively, which were genotyped by the wheat 660 K SNP array. Two hundred and four single-nucleotide polymorphisms (SNPs) were identified, among which 41(20.7%), 34 (17.2%), 22 (11.1%), and 22 (11.1%) were located on chromosomes 5A, 5B, 4B, and 5D, respectively. The genetic linkage map for the RIL population was constructed using 150 polymorphic SSR and dCAPS markers. Finally, five QTL were detected on chromosomes 5A, 5B, and 5D, designated QBB.hau-5A, QBB.hau-5B.1, QBB.hau-5B.2, QBB.hau-5D.1, and QBB.hau-5D.2, respectively. All resistance alleles were contributed by the resistant parent Yuyou1. QBB.hau-5D.1 is likely to be a new locus for black point resistance. The markers Xwmc654 and Xgwm174 linked to QBB.hau-5A and QBB.hau-5D.1, respectively, have potential utility in MAS-based breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01356-6.

Heterologous expression and characterization of ToxA1 haplotype from India and its interaction with Tsn1 for spot blotch susceptibility in spring wheat.

BACKGROUND: ToxA, a necrotrophic effector protein, is present in the genome of fungal species like Parastagnospora nodorum, Pyrenophora tritici-repentis and Bipolaris sorokiniana. Tsn1 is the sensitivity gene in the host whose presence indicates more susceptibility to ToxA carrying pathogen, and ToxA-Tsn1 interaction follows an inverse gene-for-gene relationship. METHODS AND RESULTS: The present study involved cloning and expressing the ToxA1 haplotype from B. sorokiniana. It was found that the amplicon exhibited an expected product size of 471 bp. Sequence analysis of the ToxA1 nucleotide sequence revealed the highest identity, 99.79%, with P. tritici-repentis. The protein expression analysis showed peak expression at 16.5 kDa. Phylogenetic analysis of the ToxA1 sequence from all the Bipolaris isolates formed an independent clade along with P. tritici-repentis and diverged from P. nodorum. ToxA-Tsn1 interaction was studied in 18 wheat genotypes (11 Tsn1 and 7 tsn1) at both seedling and adult stages, validating the inverse gene-for-gene relationship, as the toxin activity was highest in the K68 genotype (Tsn1) and lowest in WAMI280 (tsn1). CONCLUSION: The study indicates that the haplotype ToxA1 is prevailing in the Indian population of B. sorokiniana. It would be desirable for wheat breeders to select genotypes with tsn1 locus for making wheat resistant to spot blotch.

Identification and Fungicide Control of Bipolaris sorokiniana Causing Leaf Spot and Blight on Common Hop (Humulus lupulus) in Korea.

Hop (Humulus lupulus) is a perennial herbaceous vine belonging to the family Cannabaceae. This crop is commercially grown for the brewing industry for its bitter and aromatic flavor, as well as its antiseptic properties. In June 2021, leaf spot and blight was observed on common hop plants in Buan-gun, Jeollabuk-do, South Korea. The typical symptoms were small to large, dark-brown, necrotic lesions with yellow halos on the leaves. This study aimed to clarify the causal agent of this disease. Two fungal species, Alternaria alternata and Bipolaris sorokiniana, were isolated from the diseased leaf samples and identified by combining morphological observations and phylogenetic analysis using sequence datasets of internal transcribed spacer (ITS), Alt a1, rpb2, endoPG, and OPA10-2; and ITS, gpd, and tef1, respectively. Pathogenicity of the fungal isolates on detached leaves and living plants revealed that B. sorokiniana is the causal pathogen of this disease, while A. alternata is potentially a saprophyte. Fungicide sensitivity of the pathogen B. sorokiniana was further estimated in vitro using three classes of fungicides represented by fluxapyroxad, pyraclostrobin, and hexaconazole. The effective concentrations that inhibited 50% of spore germination (EC50) were 0.72, 1.90, and 0.68 µg ml-1, respectively. Moreover, all of these fungicides were able to control B. sorokiniana on detached common hop leaves at their recommended concentrations. In conclusion, this study reports leaf spot and blight of common hop caused by B. sorokiniana for the first time and proposes potential fungicides for this disease.

First report of melting out on creeping bentgrass caused by Bipolaris sorokiniana in China.

Creeping bentgrass (Agrostis stolonifera L.), is one of the major cool-season turfgrass species, which is widely planted in putting greens at golf courses in China (Zhou et al. 2022). In June 2022, an unknown disease with reddish-brown spots (2-5 cm in diameter) were observed on 'A4' creeping bentgrass putting greens at Longxi golf course in Bejing. As the disease progressed, the spots coalesced and formed irregular patches (15-30 cm in diameter). When looking closely, the leaves were wilting, yellowing and melting out from the foliar tips to the crowns. The disease incidence was estimated up to 10-20 % of each putting green and a total of five putting greens had similar symptoms as described above. Three to five symptomatic samples were collected from each green. Diseased leaves were cut into pieces, surface sterilized in 0.6% sodium hypochlorite (NaClO) for 1 min, washed three times with sterilized water, air dried and placed on potato dextrose agar (PDA) containing 50 mg L-1 streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25 °C for 3 days, and fungal isolates with similar morphology (irregular cultures with dark-brown back and light-brown to white surface) was consistently recovered. Pure cultures were obtained by repeat hyphal-tip transfer. The fungus did not grow well on PDA medium, radial growth was estimated 1.5 mm/d, and dark-brown colony was surrounded by light-white margin. However, it grew fast on creeping bentgrass leaf extract (CBLE) medium, CBLE medium was produced by adding 0.75 g potato powder, 5 g agar and 20 mL creeping bentgrass leaf juice (with 1 g fresh creeping bentgrass leaf) into 250 mL sterile water. The colony was sparse and light-white, and radial growth was roughly 9 mm/d on CBLE medium. Conidia were spindle-shaped, olive to brown in color, pointy or obtuse at both ends, 4 to 8 septa, and a size range of 9.85 to 20.20 × 26.26 to 45.64 µm (14.85 µm × 40.62 µm average, n = 30). Genomic DNA of two representative isolates (HH2 and HH3) was extracted, the nuclear ribosomal internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region were amplified with primers ITS1/ITS4 (White et al. 1990) and gpd1/gpd2 (Berbee et al. 1999), respectively. The ITS (OQ363182 and OQ363183) and GAPDH (OQ378336 and OQ378337) sequences were deposited in GenBank. BLAST analyses revealed that the sequences shared 100% and 99% similarity with the published ITS (CP102792) and GAPDH (CP102794) of B. sorokiniana strain LK93, respectively. To complete Koch's postulates, 3 plastic pots (15 cm height × 10 cm top diameter × 5 cm bottom diameter, three replicates for the isolate HH2) were seeded with creeping bentgrass and inoculated with a spore suspension (1×105 conidia/mL) after two months of plant growths. Healthy creeping bentgrass inoculated with distilled water served as controls. All pots were covered with plastic bags, placed in a growth chamber with a 12-h day/night cycle at 30/25°C and 90% relative humidity. Disease symptoms (yellowing and melting out leaf) were noted after 7 days. B. sorokiniana was recovered from the diseased leaves and identified morphologically and molecularly as described above. To our knowledge, this is the first report of melting out on creeping bentgrass caused by B. sorokiniana in China. The report here will provide a scientific basis for developing management strategies on this disease in the future. Additional study is needed to investigate the prevalence of the disease on putting greens from golf courses in larger regions of China.

Differential expression profiling of microRNAs and their target genes during wheat-Bipolaris sorokiniana pathosystem.

Wheat spot blotch, caused by Bipolaris sorokiniana, is a serious constraint to wheat production, reducing grain yield and consequently having severe economic impact. Several plant miRNAs have recently been discovered as regulators of gene expression involved in cellular and metabolic functions. So far reports on the roles of miRNAs in B. sorokiniana infection response of wheat are scanty. To further understand the defence mechanism of miRNAs- regulated cellular functions, we examined the expression patterns of 17 miRNAs and their targets involved in the interaction between wheat and B. sorokiniana in two contrasting wheat genotypes, Chiriya-1 and WH-147. All of the miRNAs and target genes were shown to be expressed differentially in both genotypes after B. sorokiniana infection. Seven and nine miRNAs were observed as up-regulated in the resistant genotype Chiriya-1 and the susceptible genotype WH147, respectively. Among the up-regulated miRNAs, ptc-miR901 (~ 10.21 times) accumulated the most in Chiriya-1 followed by ptc-miR1450 (~ 7.6 times) in WH-147. Furthermore, only two miRNAs, tae-miR156 and ptc-miR482c showed a complete inverse relation with their target genes, SPL and NBS-LRR, respectively. This research sheds light on the temporal differential regulation of miRNAs and their targets, which may play a role in wheat adaptation to B. sorokiniana infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01092-1.

A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

Engineering of a highly thermostable endoglucanase from the GH7 family of Bipolaris sorokiniana for higher catalytic efficiency.

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and ß-glucosidase towards complete saccharification of cellulose into glucose.

Genome-based mining of new antimicrobial meroterpenoids from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Polyketide-terpenoid hybrid compounds are one of the largest families of meroterpenoids, with great potential for drug development for resistant pathogens. Genome sequence analysis of secondary metabolite gene clusters of a phytopathogenic fungus, Bipolaris sorokiniana 11134, revealed a type I polyketide gene cluster, consisting of highly reducing polyketide synthase, non-reducing polyketide synthase, and adjacent prenyltransferase. MS- and UV-guided isolations led to the isolation of ten meroterpenoids, including two new compounds: 19-dehydroxyl-3-epi-arthripenoid A (1) and 12-keto-cochlioquinone A (2). The structures of 1-10 were elucidated by the analysis of NMR and high-resolution electrospray ionization mass spectroscopy data. Compounds 5-8 and 10 showed moderate activity against common Staphylococcus aureus and methicillin-resistant S. aureus, with minimum inhibitory concentration (MIC) values of 12.5-100 µg/mL. Compound 5 also exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5-25 µg/mL. The biosynthetic gene cluster of the isolated compounds and their putative biosynthetic pathway are also proposed. KEY POINTS: • Ten meroterpenoids were identified from B. sorokiniana, including two new compounds. • Cochlioquinone B (5) showed activity against MRSA and synergistic activity against C. albicans. • The biosynthetic gene cluster and biosynthetic pathway of meroterpenoids are proposed. • Genome mining provided a new direction to uncover the diversity of meroterpenoids.

Quantitative Trait Loci Mapping for Spot Blotch Resistance in Two Biparental Mapping Populations of Bread Wheat.

Spot blotch (SB), caused by Bipolaris sorokiniana, is a major fungal disease of wheat in South Asia and South America. Two biparental mapping populations with 232 F2:7 progenies each were generated, with CIMMYT breeding lines CASCABEL and KATH as resistant parents and CIANO T79 as the common susceptible parent. The two populations were evaluated for field SB resistance in CIMMYT's Agua Fria station for three consecutive cropping seasons, with artificial inoculation. Genotyping was done with the DArTseq platform and approximately 1,500 high quality and nonredundant markers were used for quantitative trait loci (QTL) mapping. In both populations, a major QTL was found on chromosome 5A in the Vrn-A1 region, explaining phenotypic variations of 13.5 to 25.9%, which turned up to be less- or nonsignificant when days to heading and plant height were used as covariates in the analysis, implying a disease escape mechanism. Another major QTL was located on chromosome 5B in CASCABEL, accounting for 8.9 to 21.4% of phenotypic variation. Minor QTL were found on 4A and 4B in CASCABEL; 1B, 4B, and 4D in KATH; and 1B, 2B, and 4B in CIANO T79. Through an analysis of QTL projection onto the IWGSC Chinese Spring reference genome, the 5B QTL in CASCABEL was mapped in the Sb2 region, delimited by the single nucleotide polymorphism marker wsnp_Ku_c50354_55979952 and the simple sequence repeat marker gwm213, with a physical distance of about 14 Mb to the Tsn1 locus.

ToxA-Tsn1 Interaction for Spot Blotch Susceptibility in Indian Wheat: An Example of Inverse Gene-for-Gene Relationship.

The ToxA-Tsn1 system is an example of an inverse gene-for-gene relationship. The gene ToxA encodes a host-selective toxin (HST) which functions as a necrotrophic effector and is often responsible for the virulence of the pathogen. The genomes of several fungal pathogens (e.g., Pyrenophora tritici-repentis, Parastagonospora nodorum, and Bipolaris sorokiniana) have been shown to carry the ToxA gene. Tsn1 is a sensitivity gene in the host, whose presence generally helps a ToxA-positive pathogen to cause spot blotch in wheat. Cultivars lacking Tsn1 are generally resistant to spot blotch; this resistance is attributed to a number of other known genes which impart resistance in the absence of Tsn1. In the present study, 110 isolates of B. sorokiniana strains, collected from the ME5A and ME4C megaenvironments of India, were screened for the presence of the ToxA gene; 77 (70%) were found to be ToxA positive. Similarly, 220 Indian wheat cultivars were screened for the presence of the Tsn1 gene; 81 (36.8%) were found to be Tsn1 positive. When 20 wheat cultivars (11 with Tsn1 and 9 with tsn1) were inoculated with ToxA-positive isolates, seedlings of only those carrying the Tsn1 allele (not tsn1) developed necrotic spots surrounded by a chlorotic halo. No such distinction between Tsn1 and tsn1 carriers was observed when adult plants were inoculated. This study suggests that the absence of Tsn1 facilitated resistance against spot blotch of wheat. Therefore, the selection of wheat genotypes for the absence of the Tsn1 allele can improve resistance to spot blotch.

Assessing Genetic Resistance in Wheat to Black Point Caused by Six Fungal Species in the Yellow and Huai Wheat Area of China.

The most effective and environmentally sustainable method for controlling black point disease of wheat (Triticum aestivum L.) is to plant resistant cultivars. To identify sources of resistance to black point, 165 selected cultivars/lines were inoculated with isolates of six fungal species (Bipolaris sorokiniana, Alternaria alternata, Fusarium equiseti, Exserohilum rostratum, Epicoccum sorghinum, and Curvularia spicifera) known to cause black point in wheat using spore suspensions under controlled field conditions in 2016 and 2017. Inoculation of the isolates significantly increased the incidence of black point in the cultivars/lines compared with those grown under natural field conditions (NFC). The disease incidence of plants inoculated with B. sorokiniana and E. rostratum was 15.5% and 18.8% in 2016, and 20.4% and 23.0% in 2017, whereas those under NFC were 5.7% (2016) and 1.5% (2017), respectively. Furthermore, disease symptoms varied with pathogen. Among the 165 cultivars/lines tested, 3.6%, 50.9%, 60.0%, 1.8%, 47.3%, and 58.8% were resistant to B. sorokiniana, A. alternata, F. equiseti, E. rostratum, E. sorghinum, and C. spicifera, respectively. In addition, we identified one line ('SN530070') resistant to black point caused by all six pathogens. This is the first study to assess resistance to wheat black point caused by six fungal species under controlled conditions. The black point-resistant cultivars/lines could be useful in breeding and also in research on the mechanisms of resistance to black point.

Genome Sequence Resource for Pathogen Bipolaris sorokiniana Shoemaker GN1 Causing Spot Blotch of Barley (Hordeum vulgare L.).

Spot blotch, caused by fungal pathogen Bipolaris sorokiniana Shoemaker, is one of the most frequent diseases affecting barley-growing regions worldwide. In this study, we reported the genome sequence of the highly virulent B. sorokiniana strain GN1 using the Illumina HiSeq 4000 platform. In total, 57 million 150-nucleotide paired-end clean reads were obtained and assembled into 96 scaffolds with an estimated genome size of 34.33 Mb. Furthermore, we identified genes that may be associated with strain-specific virulence and performed phylogenetic analysis of GN1 with five other Bipolaris spp. These results for GN1 will provide important information in understanding its molecular underpinning of pathogenicity and help identify novel sources of genetic resistance for improving disease resistance in barley.

The Cysteine-Rich Repeat Protein TaCRR1 Participates in Defense against Both Rhizoctonia cerealis and Bipolaris sorokiniana in Wheat.

The domain of unknown function 26 (DUF26), harboring a conserved cysteine-rich motif (C-X8-C-X2-C), is unique to land plants. Several cysteine-rich repeat proteins (CRRs), belonging to DUF26-containing proteins, have been implicated in the defense against fungal pathogens in ginkgo, cotton, and maize. However, little is known about the functional roles of CRRs in the important staple crop wheat (Triticum aestivum). In this study, we identified a wheat CRR-encoding gene TaCRR1 through transcriptomic analysis, and dissected the defense role of TaCRR1 against the soil-borne fungi Rhizoctonia cerealis and Bipolaris sorokiniana, causal pathogens of destructive wheat diseases. TaCRR1 transcription was up-regulated in wheat towards B. Sorokiniana or R. cerealis infection. The deduced TaCRR1 protein contained a signal peptide and two DUF26 domains. Heterologously-expressed TaCRR1 protein markedly inhibited the mycelia growth of B. sorokiniana and R. cerealis. Furthermore, the silencing of TaCRR1 both impaired host resistance to B. sorokiniana and R. cerealis and repressed the expression of several pathogenesis-related genes in wheat. These results suggest that the TaCRR1 positively participated in wheat defense against both B. sorokiniana and R. cerealis through its antifungal activity and modulating expression of pathogenesis-related genes. Thus, TaCRR1 is a candidate gene for improving wheat resistance to B. sorokiniana and R. cerealis.

Transcriptomics analysis of propiconazole-treated Cochliobolus sativus reveals new putative azole targets in the plant pathogen.

Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is a filamentous fungus from the class Dothideomycetes. It is a pathogen of cereals including wheat and barley, and causes foliar spot blotch, root rot, black point on grains, head blight, leaf blight, and seedling blight diseases. Annual yields of these economically important cereals are severely reduced due to this pathogen attack. Evolution of fungicide resistant pathogen strains, availability of a limited number of potent antifungal compounds, and their efficacy are the acute issues in field management of phytopathogenic fungi. Propiconazole is a widely used azole fungicide to control the disease in fields. The known targets of azoles are the demethylase enzymes involved in ergosterol biosynthesis. Nonetheless, azoles have multiple modes of action, some of which have not been explored yet. Identifying the off-target effects of fungicides by dissecting gene expression profiles in response to them can provide insights into their modes of action and possible mechanisms of fungicide resistance. Moreover it can also reveal additional targets for development of new fungicides. Hence, we analyzed the global gene expression profile of C. sativus on exposure to sub-lethal doses of propiconazole in a time series. The gene expression patterns were confirmed using quantitative reverse transcriptase PCR (qRT-PCR). This study revealed overexpression of target genes from the sterol biosynthesis pathway supporting the reported mode of resistance against azoles. In addition, some new potential targets have also been identified, which could be explored to develop new fungicides and plant protection strategies.

FGB1 and WSC3 are in planta-induced ß-glucan-binding fungal lectins with different functions.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in ß-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain ß1-3-glucan but has no affinity for shorter ß1-3- or ß1-6-linked glucose oligomers. Comparative analysis with the previously identified ß-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require ß1-6-linked glucose for efficient binding to branched ß1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two ß-glucan-binding lectins. Our results highlight the importance of the ß-glucan cell wall component in plant-fungus interactions and the potential of ß-glucan-binding lectins as specific detection tools for fungi in vivo.

Genome- and MS-based mining of antibacterial chlorinated chromones and xanthones from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Halogen substituents are important for biological activity in many compounds. Genome-based mining of halogenase along with its biosynthetic gene cluster provided an efficient approach for the discovery of naturally occurring organohalogen compounds. Analysis of the genome sequence of a phytopathogenic fungus Bipolaris sorokiniana 11134 revealed a polyketide gene cluster adjacent to a flavin-dependent halogenase capable of encoding halogenated polyketides, which are rarely reported in phytopathogenic fungi. Furthermore, MS- and UV-guided isolation and purification led to the identification of five chlorine-containing natural products together with seven other chromones and xanthones. Two of the chlorinated compounds and four chromones are new compounds. Their structures were elucidated by NMR spectroscopic analysis and HRESIMS data. The biosynthetic gene clusters of isolated compounds and their putative biosynthetic pathway are also proposed. One new chlorinated compound showed activity against Staphylococcus aureus, methicillin-resistant S. aureus, and three clinical-resistant S. aureus strains with a shared minimum inhibitory concentration (MIC) of 12.5 µg/mL. Genome-based mining of halogenases combined with high-resolution MS- and UV-guided identification provides an efficient approach to discover new halogenated natural products from microorganisms.

Disclosure of the Molecular Mechanism of Wheat Leaf Spot Disease Caused by Bipolaris sorokiniana through Comparative Transcriptome and Metabolomics Analysis.

Wheat yield is greatly reduced because of the occurrence of leaf spot diseases. Bipolaris sorokiniana is the main pathogenic fungus in leaf spot disease. In this study, B. sorokiniana from wheat leaf (W-B. sorokiniana) showed much stronger pathogenicity toward wheat than endophytic B. sorokiniana from Pogostemon cablin (P-B. sorokiniana). The transcriptomes and metabolomics of the two B. sorokiniana strains and transcriptomes of B. sorokiniana-infected wheat leaves were comparatively analyzed. In addition, the expression levels of unigenes related to pathogenicity, toxicity, and cell wall degradation were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Results indicated that pathogenicity-related genes, especially the gene encoding loss-of-pathogenicity B (LopB) protein, cell wall-degrading enzymes (particularly glycosyl hydrolase-related genes), and killer and Ptr necrosis toxin-producing related unigenes in the W-B. sorokiniana played important roles in the pathogenicity of W-B. sorokiniana toward wheat. The down-regulation of cell wall protein, photosystem peptide, and rubisco protein suggested impairment of the phytosynthetic system and cell wall of B. sorokiniana-infected wheat. The up-regulation of hydrolase inhibitor, NAC (including NAM, ATAF1 and CUC2) transcriptional factor, and peroxidase in infected wheat tissues suggests their important roles in the defensive response of wheat to W-B. sorokiniana. This is the first report providing a comparison of the transcriptome and metabolome between the pathogenic and endophytic B. sorokiniana strains, thus providing a molecular clue for the pathogenic mechanism of W-B. sorokiniana toward wheat and wheat's defensive response mechanism to W-B. sorokiniana. Our study could offer molecular clues for controlling the hazard of leaf spot and root rot diseases in wheat, thus improving wheat yield in the future.

Biosynthesis of Phenylamide Phytoalexins in Pathogen-Infected Barley.

Phytoalexins are inducible antimicrobial metabolites in plants, and have been indicated to be important for the rejection of microbial infection. HPLC analysis detected the induced accumulation of three compounds 1-3 in barley (Hordeum vulgare) roots infected by Fusarium culmorum, the causal agent of Fusarium root rot. Compounds 1-3 were identified as cinnamic acid amides of 9-hydroxy-8-oxotryptamine, 8-oxotryptamine, and (1H-indol-3-yl)methylamine, respectively, by spectroscopic analysis. Compounds 1 and 2 had been previously reported from wheat, whereas 3 was an undescribed compound. We named 1-3 as triticamides A-C, respectively, because they were isolated from barley and wheat, which belong to the Triticeae tribe. These compounds showed antimicrobial activities, indicating that triticamides function as phytoalexins in barley. The administration of deuterium-labeled N-cinnamoyl tryptamine (CinTry) to barley roots resulted in the effective incorporation of CinTry into 1 and 2, which suggested that they were synthesized through the oxidation of CinTry. Nine putative tryptamine hydroxycinnamoyl transferase (THT)-encoding genes (HvTHT1-HvTHT9) were identified by database search on the basis of homology to known THT gene sequences from rice. Since HvTHT7 and HvTHT8 had the same sequences except one base, we measured their expression levels in total by RT-qPCR. HvTHT7/8 were markedly upregulated in response to infection by F. culmorum. The HvTHT7 and HvTHT8 enzymes preferred cinnamoyl- and feruloyl-CoAs as acyl donors and tryptamine as an acyl acceptor, and (1H-indol-3-yl)methylamine was also accepted as an acyl acceptor. These findings suggested that HvTHT7/8 are responsible for the induced accumulation of triticamides in barley.

Bipolenins K-N: New sesquiterpenoids from the fungal plant pathogen Bipolaris sorokiniana.

Chemical investigation of the barley and wheat fungal pathogen Bipolaris sorokiniana BRIP10943 yielded four new sativene-type sesquiterpenoid natural products, bipolenins K-N (1-4), together with seven related known analogues (5-11), and a sesterterpenoid (12). Their structures were determined by detailed analysis of spectroscopic data, supported by TDDFT calculations and comparison with previously reported analogues. These compounds were evaluated for their phytotoxic activity against wheat seedlings and wheat seed germination. The putative biosynthetic relationships between the isolated sesquiterpenoids were also explored.

Resistance to Spot Blotch in Two Mapping Populations of Common Wheat Is Controlled by Multiple QTL of Minor Effects.

Spot blotch (SB) is an important fungal disease of wheat in South Asia and South America. Host resistance is regarded as an economical and environmentally friendly approach of controlling SB, and the inheritance of resistance is mostly quantitative. In order to gain a better understanding on the SB resistance mechanism in CIMMYT germplasm, two bi-parental mapping populations were generated, both comprising 232 F2:7 progenies. Elite CIMMYT breeding lines, BARTAI and WUYA, were used as resistant parents, whereas CIANO T79 was used as susceptible parent in both populations. The two populations were evaluated for field SB resistance at CIMMYT's Agua Fria station for three consecutive years, from the 2012⁻2013 to 2014⁻2015 cropping seasons. Phenological traits like plant height (PH) and days to heading (DH) were also determined. Genotyping was performed using the DArTSeq genotyping-by-sequencing (GBS) platform, and a few D-genome specific SNPs and those for phenological traits were integrated for analysis. The most prominent quantitative trait locus (QTL) in both populations was found on chromosome 5AL at the Vrn-A1 locus, explaining phenotypic variations of 7⁻27%. Minor QTL were found on chromosomes 1B, 3A, 3B, 4B, 4D, 5B and 6D in BARTAI and on chromosomes 1B, 2A, 2D and 4B in WUYA, whereas minor QTL contributed by CIANO T79 were identified on chromosome 1B, 1D, 3A, 4B and 7A. In summary, resistance to SB in the two mapping populations was controlled by multiple minor QTL, with strong influence from Vrn-A1.