QTL Mapping for Adult-Plant Resistance to Leaf Rust in Italian Wheat Cultivar Libellula.

Wheat leaf rust (Lr), which is caused by Puccinia triticina Eriks. (Pt), is one of the most important wheat diseases affecting wheat production globally. Using resistant wheat cultivars is the most economical and environmentally friendly way to control leaf rust. The Italian wheat cultivar Libellula has demonstrated good resistance to Lr in field studies. To identify the genetic basis of Lr resistance in 'Libellula', 248 F6 recombinant inbred lines from the cross 'Libellula'/'Huixianhong' was phenotyped for Lr severity in seven environments: the 2014/2015, 2016/2017, 2017/2018, and 2018/2019 cropping seasons at Baoding, Hebei Province, and the 2016/2017, 2017/2018, and 2018/2019 crop seasons at Zhoukou, Henan Province. Bulked segregant analysis and simple sequence repeat markers were then used to identify the quantitative trait loci (QTLs) for Lr adult-plant resistance in the population. Six QTLs were consequently detected and designated as QLr.hebau-1AL and QLr.hebau-1AS that were presumed to be new and QLr.hebau-1BL, QLr.hebau-3AL, QLr.hebau-4BL, and QLr.hebau-7DS that were identified at similar physical positions as previously reported QTLs. Based on chromosome positions and molecular marker tests, QLr.hebau-1BL and QLr.hebau-7DS share similar flanking markers with Lr46 and Lr34, respectively. Lr46 and Lr34 are race nonspecific adult plant resistance (APR) genes for leaf rust and stripe rust and powdery mildew. QLr.hebau-4BL showed multiple disease resistance to leaf rust, stripe rust, Fusarium head blight, and powdery mildew. The QTL identified in this study, as well as their closely linked markers, may potentially be used in marker-assisted selection in wheat breeding.

TaTLP1 interacts with TaPR1 to contribute to wheat defense responses to leaf rust fungus.

Thaumatin-like proteins (TLPs), which are defined as pathogenesis-related protein family 5 (PR5) members, are common plant proteins involved in defense responses and confer antifungal activity against many plant pathogens. Our earlier studies have reported that the TaTLP1 gene was isolated from wheat and proved to be involved in wheat defense in response to leaf rust attack. The present study aims to identify the interacting proteins of TaTLP1 and characterize the role of the interaction between wheat and Puccinia triticina (Pt). Pull-down experiments designed to isolate the molecular target of TaTLP1 in tobacco resulted in the identification of TaPR1, a pathogenesis-related protein of family 1, and the interaction between TaTLP1 and TaPR1 was confirmed by yeast two-hybrid experiments (Y2H), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP). In vitro, TaTLP1 and TaPR1 together increased antifungal activity against Pt. In vivo, the disease resistance phenotype, histological observations of fungal growth and host responses, and accumulation of H2O2 in TaTLP1-TaPR1 in co-silenced plants indicated that co-silencing significantly enhanced wheat susceptibility compared to single knockdown TaTLP1 or TaPR1 plants. The accumulation of reactive oxygen species (ROS) was significantly reduced in co-silenced plants compared to controls during Pt infection, which suggested that the TaTLP1-TaPR1 interaction positively modulates wheat resistance to Pt in an ROS-dependent manner. Our findings provide new insights for understanding the roles of two different PRs, TaTLP1 and TaPR1, in wheat resistance to leaf rust.

First Report of Rust Caused by Tranzschelia discolor on Peach Leaves in Shenzhou, China.

Peach (Prunus persica [L.] Batsch) as an economically important fruit tree is widely cultivated in Shenzhou, China. In September 2021, peach rust was observed in the peach tree in Shenzhou City, Hebei Province (lat. 38°02'56'' N, long. 115°54'57'' E, altitude 22 m). We investigated a peach orchard with a planting area of 1334 m2, where a total of 162 peach trees were planted, and found that about 10% of peach trees exhibited severe disease symptoms. The leaves of infected plant developed 100% disease symptoms, in which 50% of the infected leaves showed about 10 small pale-yellow spots on the front of leaves and reddish-brown pustules on the corresponding abaxial surface of leaves. Urediniospores varied from obovoid to clavate in shape, sometimes in irregular shape. They were orange-brown, echinulate near base with spines smaller towards apex and often smooth at apex, with germ pores 3-4 at equator, size ranging from 25.4 to 38.6 × 10.1 to 18.7 µm (n=100), and with wall 1 to 1.5µm thick at sides and 5-7 µm thick at apex. Golden capitate paraphyses were present, ranging from 25 to 40 µm in length, with a head in diameter of 12 to 14 µm and a tail in width of 5.2 to 6.5 µm. Based on the rust morphological characters, this pathogen was primarily identified as Tranzschelia discolor (Fuckel) Tranzschel & Litv. (Hiratsuka et al. 1992). For molecular identification, total DNA was extracted from 2 isolates, respectively, and the internal transcribed spacer (ITS) region was PCR-amplified using the primer set ITS5-u and ITS4-u (Pfunder et al. 2001). Obtained sequences were compared with sequences in the GenBank repository using BLAST algorithm. BLAST showed a 100% sequence identify to T. discolor (accession nos. AB097449、MT786217、KU712078、KY764179、MH599069). The sequence has been deposited in GenBank with (accession NO. ON950745 and ON950747). Thus, combining morphological observations and molecular identification, the isolate was identified as T. discolor. The pathogenicity was verified by inoculating the abaxial surface of peach leaves with a suspension of 1 × 106 urediniospores/ml. Peach leaves sprayed with sterile water were used as controls. The inoculated peach trees were placed in a greenhouse at 20°C under dark for 24 hours and maintained at 100% relative humidity to promote disease development. Next, the peach trees were grown in a greenhouse at 20°C with a 12 h day length and symptoms were observed on the leaves 14 days after inoculation. In contrast, the control leaves were asymptomatic. Previous studies reported that peach rust occurred in Oman, Korea and Brazil was caused by T. discolor. (Deadman M L, et al.2007, Shin, H D, et al. 2019, Vidal G S, et al. 2021). To our knowledge, this is the first report of T. discolor as a causal agent causing peach leaf rust in Northern China, which will enable us to rapidly diagnose this disease, identify the occurrence of this disease and develop adequate management strategies to control it in China.

Comparative Evaluation of the Phytochemical Profiles and Antioxidant Potentials of Olive Leaves from 32 Cultivars Grown in China.

Olives (Olea europaea L.) are a significant part of the agroindustry in China. Olive leaves, the most abundant by-products of the olive and olive oil industry, contain bioactive compounds that are beneficial to human health. The purpose of this study was to evaluate the phytochemical profiles and antioxidant capacities of olive leaves from 32 cultivars grown in China. A total of 32 phytochemical compounds were identified using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, including 17 flavonoids, five iridoids, two hydroxycinnamic acids, six triterpenic acids, one simple phenol, and one coumarin. Specifically, olive leaves were found to be excellent sources of flavonoids (4.92-18.29 mg/g dw), iridoids (5.75-33.73 mg/g dw), and triterpenic acids (15.72-35.75 mg/g dw), and considerable variations in phytochemical content were detected among the different cultivars. All tested cultivars were classified into three categories according to their oil contents for further comparative phytochemicals assessment. Principal component analysis indicated that the investigated olive cultivars could be distinguished based upon their phytochemical profiles and antioxidant capacities. The olive leaves obtained from the low-oil-content (<16%) cultivars exhibited higher levels of glycosylated flavonoids and iridoids, while those obtained from high-oil-content (>20%) cultivars contained mainly triterpenic acids in their compositions. Correspondingly, the low-oil-content cultivars (OL3, Frantoio selection and OL14, Huaou 5) exhibited the highest ABTS antioxidant activities (758.01 ± 16.54 and 710.64 ± 14.58 mg TE/g dw, respectively), and OL9 (Olea europaea subsp. Cuspidata isolate Yunnan) and OL3 exhibited the highest ferric reducing/antioxidant power assay values (1228.29 ± 23.95 mg TE/g dw and 1099.99 ± 14.30 mg TE/g dw, respectively). The results from this study may be beneficial to the comprehensive evaluation and utilization of bioactive compounds in olive leaves.

TaNAC35 acts as a negative regulator for leaf rust resistance in a compatible interaction between common wheat and Puccinia triticina.

NAC (NAM, AFAT1/2, and CUC2) transcription factors play important roles in plant growth and in resistance to abiotic and biotic stresses. Here, we show that the TaNAC35 gene negatively regulates leaf rust resistance in the wheat line Thatcher + Lr14b (TcLr14b) when challenged with a virulent isolate of Puccinia triticina (Pt). The TaNAC35 gene was cloned from this line, and blastp results showed that its open reading frame (ORF) was 96.16% identical to the NAC35-like sequence reported from Aegilops tauschii, and that it encoded a protein with 387 amino acids (aa) including a conserved NAM domain with 145 aa at the N-terminal alongside the transcriptional activation domain with 220 aa in the C-terminal. Yeast-one-hybrid analysis proved that the C-terminal of the TaNAC35 protein was responsible for transcriptional activation. A 250-bp fragment from the 3'-end of this target gene was introduced to a BSMV-VIGS vector and used to infect the wheat line Thatcher + Lr14b (TcLr14b). The BSMV-VIGS/TaNAC35-infected plant material showed enhanced resistance (infection type "1") to Pt pathotype THTT, which was fully virulent (infection type "4") on BSMV-VIGS only infected TcLr14b plants. Histological studies showed that inhibition of TaNAC35 reduced the formation of haustorial mother cells (HMC) and mycelial growth, implying that the TaNAC35 gene plays a negative role in the response of TcLr14b to Pt pathotype THTT. These results provide molecular insight into the interaction between Pt and its wheat host, and identify a potential target for engineering resistance in wheat to this damaging pathogen.

Identification and Predictions Regarding the Biosynthesis Pathway of Polyene Macrolides Produced by Streptomyces roseoflavus Men-myco-93-63.

A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC50) of 2.05 to 7.09 µg/ml and 90% effective concentrations (EC90) of 4.32 to 54.45 µg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments.IMPORTANCEStreptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.

Genome-wide association mapping of leaf rust and stripe rust resistance in wheat accessions using the 90K SNP array.

KEY MESSAGE: A genome-wide association analysis identified diverse loci for seedling and adult plant resistance to leaf rust and stripe rust. KASP markers were developed and validated for marker-assisted selection. Wheat leaf rust and stripe rust cause significant losses in many wheat producing regions worldwide. The objective of this study was to identify chromosome regions conferring resistance to both leaf rust and stripe rust at the seedling and adult plant stages. A diversity panel of 268 wheat lines, including 207 accessions from different wheat growing regions in China, and 61 accessions from foreign countries, were evaluated for leaf rust response at seedling stage using eight Chinese Puccinia triticina pathotypes, and also tested for leaf rust and stripe rust at adult plant stage in multiple field environments. The panel was genotyped with the Wheat 90 K Illumina iSelect SNP array. Genome-wide association mapping (GWAS) was performed using the mixed linear model (MLM). Twenty-two resistance loci including the known Lr genes, Lr1, Lr26, Lr3ka, LrZH22, and 18 potentially new loci were identified associated with seedling resistance, explaining 4.6 to 25.2% of the phenotypic variance. Twenty-two and 23 adult plant resistance (APR) QTL associated with leaf and stripe rust, respectively, were identified at adult stage, explaining 4.2-11.5% and 4.4-9.7% of the phenotypic variance. Among them, QLr-2BS was the potentially most valuable all-stage resistance gene. Seven and six consistent APR QTL were identified in multiple environments including best linear unbiased prediction (BLUP) data, respectively. Comparison with previously mapped resistance loci indicated that three of the seven leaf rust resistance APR QTL, and two of the six stripe rust resistance APR QTL were new. Four potentially pleiotropic APR QTL, including Lr46/Yr29, QLr-2AL.1/QYr-2AL.1, QLr-2AL.2/QYr-2AL.2, and QLr-5BL/QYr-5BL.1, were identified. Twelve associated SNPs were converted into kompetitive allele-specific PCR (KASP) markers and verified in bi-parental populations. The study reports genetic loci conferring resistance to both diseases, and the closely linked markers should be applicable for marker-assisted wheat breeding.

Culture-independent analysis of the bacterial community in Chinese fermented vegetables and genomic analysis of lactic acid bacteria.

Six different fermented vegetables were collected from Zhejiang Province, China, to explore the associated bacterial community using a high-throughput sequencing platform. A total of 24 phyla, 274 families and 569 genera were identified from 6 samples. Firmicutes and Proteobacteria were the main phyla in all of the samples. Brevibacterium was the major genus in Xiaoshan pickled radish. Lactobacillus-related genera and Vibrio were the major genera in fermented potherb mustard and its brine. Enterobacter and Cobetia were the major genera in fermented radish and its brine. Chromohalobacter was the major genus in the tuber mustard. These results indicated clear differences were there between the bacterial genera present in Xiaoshan pickled radish, fermented potherb mustard, fermented radish, and tuber mustard. This demonstrated the possible influences of raw materials and manufacturing processes. Furthermore, a large number of lactic acid bacteria were isolated and identified by culture-dependent and 16S rRNA gene sequence analysis, which accounted for more than 68% of all the isolates. In addition, whole-genome analysis of Levilactobacillus suantsaii, Latilactobacillus sakei subsp. sakei, and Weissella cibaria showed that they had large numbers of genes associated with carbohydrate metabolism. This may explain why these three bacterial strains can grow in fermented vegetable environments.

Synergistic Action of Commercially Available Fungicides for Protecting Wheat from Common Root Rot Caused by Bipolaris sorokiniana in China.

Wheat (Triticum aestivum) common root rot (CRR) caused by predominant fungal pathogen Bipolaris sorokiniana occurs in all wheat-growing regions worldwide and is difficult to control. In this study, the efficacy of eight fungicides against Bipolaris sorokiniana was examined in in vitro assays, and we determined that the combined application of two fungicides significantly inhibits the growth of fungal mycelium. Half of the maximal effective concentration of a mixture containing fludioxonil and difenoconazole in the ratio 1:4 was 0.0372 mg/liter, and the cotoxicity coefficient was 160.14. Under an environmentally controlled pot assay, seed treatment with the mixture of fludioxonil and difenoconazole in the 1:4 ratio demonstrated the best control efficiency at seedling and adult stages, respectively. The best synergistic mixture on seed treatment was assessed in a 2-year field experiment at Hebei, China. The best control efficacy achieved at the seedling and adult stages was 82.65% and 68.48%, respectively. Overall, the in vitro mycelial growth inhibition assay and controlled-environment and field studies indicated that the synergistic action of a mixture of fludioxonil and difenoconazole provides effective control against wheat CRR. These findings highlight the potential application of the fungicide combination for controlling CRR and reducing the selection pressure on fungal pathogens by lessening the use of various fungicides in the field.

Citrobacter freundii causing Ginger (Zingiber officinale Rosc.) Rot in Tangshan, China.

Ginger (Zingiber officinale Rosc.) is an important economic crop and its rhizome can be used as seasoning agent and traditional medicine in China. During July 2018 and 2019, decay symptoms occurred in the ginger planting area of Tangshan City, Hebei Province, with incidence rates of 15%～20%. The pathogen infected the rhizomes and leaves. The symptoms included leaves chlorosis and gradually wilting, even the whole plant wilted, the rhizome became soft and presented light brown maceration. In serious cases, the interior of rhizome was completely eroded, gray-white juice overflowing the epidermis, and with foul smell. The rhizome surfaces of ginger plants were disinfected with 1% NaOCl, and colonies were isolated and purified on nutrient agar (NA) solid medium by streaking. Eight isolates were obtained from 15 diseased tissue samples. Further morphological, physiological and biochemical identification of the pure cultured bacteria were carried out. Three strains of bacteria were picked for further analysis. All of the three strains were gram-negative, short rod-shaped，nonmotile bacillus. Colonies were round and milky yellow, smooth raised, and moist after incubation at 28°C for 24h on NA. Physiological and biochemical test results showed that strains were facultatively anaerobic, negative for indole, methyl red, the Voges-Prauskauer test (V-P) and urease; positive for glucose, sucrose, sorbitol, inositol, mannitol, citrate utilization and hydrogen sulfide production; gelatin liquefaction. A typical hypersensitive reaction was induced on 12-week-old tobacco (Nicotiana benthamiana) leaves, which were inoculated by injecting suspensions of the isolated strain (108 CFU/mL) at 25 ℃ after 24h. These characteristics were consistent with Citrobacter freundii (Werkman and Gillen 1932). To further assess the identity of the strains, the genomic DNA was extracted from one bacterium（JXJ4）. The partial 16S rRNA region (Lane 1991) and specific rpoB and gyrB genes (Mollet et al. 1997, Brady et al. 2013) were amplified and sequenced with primers 27F/1492R, CM7/CM31b and UP1f/UP2r, respectively. The obtained 16S, rpoB and gyrB sequences (GenBank accession MN148645, MN158728 and MW199734) of the isolate showed 99.93%, 99.51% and 99.82% identity to the corresponding sequences of C. freundii in GenBank (CP024679.1, CP024677.1 and KM509081.1). Maximum likelihood analysis was performed, and the phylogenetic tree clustered with C. freundii (MEGAX, Bootstrap n=1000). The pathogenicity of the isolates was tested on ginger plants and rhizomes tissue. The bacterial suspensions (108 CFU/mL) of three isolates were injected into the basal stem and rhizomes center of 9 healthy ginger seedlings respectively, and Control groups were treated with sterile water. The inoculated plants were kept in a moist chamber (28°C, 16-h light and 8-h dark period) and ginger rhizomes were placed in the incubator (30°C, 16-h light and 8-h dark period). Seven days after inoculation, the ginger tubers showed symptoms of decay, and 20 to 25 days later, the ginger plant leaves browned and died. The pathogenicity test was repeated 4 times and all controls were healthy. Pathogens were reisolated from symptomatic plants and rhizomes and identified as C. freundii based on the morphological, biochemical and molecular methods described previously, fulfilling Koch's hypothesis. To our knowledge, this is the first report of ginger rot caused by C. freundii in China.

WRKY Transcription Factors Shared by BTH-Induced Resistance and NPR1-Mediated Acquired Resistance Improve Broad-Spectrum Disease Resistance in Wheat.

In Arabidopsis, both pathogen invasion and benzothiadiazole (BTH) treatment activate the nonexpresser of pathogenesis-related genes 1 (NPR1)-mediated systemic acquired resistance, which provides broad-spectrum disease resistance to secondary pathogen infection. However, the BTH-induced resistance in Triticeae crops of wheat and barley seems to be accomplished through an NPR1-independent pathway. In the current investigation, we applied transcriptome analysis on barley transgenic lines overexpressing wheat wNPR1 (wNPR1-OE) and knocking down barley HvNPR1 (HvNPR1-Kd) to reveal the role of NPR1 during the BTH-induced resistance. Most of the previously designated barley chemical-induced (BCI) genes were upregulated in an NPR1-independent manner, whereas the expression levels of several pathogenesis-related (PR) genes were elevated upon BTH treatment only in wNPR1-OE. Two barley WRKY transcription factors, HvWRKY6 and HvWRKY70, were predicted and further validated as key regulators shared by the BTH-induced resistance and the NPR1-mediated acquired resistance. Wheat transgenic lines overexpressing HvWRKY6 and HvWRKY70 showed different degrees of enhanced resistance to Puccinia striiformis f. sp. tritici pathotype CYR32 and Blumeria graminis f. sp. tritici pathotype E20. In conclusion, the transcriptional changes of BTH-induced resistance in barley were initially profiled, and the identified key regulators would be valuable resources for the genetic improvement of broad-spectrum disease resistance in wheat.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Puccinia triticina pathotypes THTT and THTS display complex transcript profiles on wheat cultivar Thatcher.

BACKGROUND: Wheat leaf rust is an important disease worldwide. Understanding the pathogenic molecular mechanism of Puccinia triticina Eriks. (Pt) and the inconstant toxic region is critical for managing the disease. The present study aimed to analyze the pathogenic divergence between Pt isolates. RESULTS: Total RNA was extracted from the wheat cultivar Thatcher infected by two Pt isolates, Tc361_1 (THTT) and Tc284_2 (THTS), at 144 h post inoculation (hpi). The mRNA was then sequenced, and a total of 2784 differentially expressed genes (DEGs) were detected. Forty-five genes were specifically expressed in THTT; these genes included transcription initiation factors and genes with transmembrane transporter activity and other genes. Twenty-six genes were specifically expressed in THTS, including genes with GTPase activity, ABC transporters and other genes. Fifty-four differentially expressed candidate effectors were screened from the two isolates. Two candidate effectors were chosen and validated on tobacco, and the results showed that they could inhibit necrosis induced by BAX. qRT-PCR of 12 significant DEGs was carried out to validate that the results are similar to those of RNA-seq at 144 hpi, to show the expression levels of these DEGs in the early stage and to elucidate the differences in expression between the two Pt pathotypes. CONCLUSION: The results obtained in this study showed that although the two pathotypes of THTT and THTS contribute similar virulence to wheat, there are a large number of genes participate in the interaction with the susceptible wheat cultivar Thatcher, and revealed the pathogenicity of rust is very complicated.

Mapping of Stripe Rust and Leaf Rust Resistance Quantitative Trait Loci in the Chinese Spring Wheat Line Mianyang351-15.

Stripe rust and leaf rust cause wheat yield losses of up to 70% worldwide. The employment of resistant cultivars is the major method to reduce losses from these diseases. The objective of this study was to detect quantitative trait loci (QTL) for stripe rust and leaf rust resistance in 150 F6 recombinant inbred lines (RIL) derived from a cross between Mianyang351-15 and Zhengzhou 5389. Both parents and the RIL population were genotyped with the Wheat55K single nucleotide polymorphism (SNP) array and simple sequence repeat markers, and phenotyped for stripe rust severity at Mianyang in Sichuan Province and Baoding in Hebei Province, and for leaf rust severity at Zhoukou in Henan Province and at Baoding in 2014 to 2017 cropping seasons. Seven and four QTL all contributed from Mianyang351-15 were identified for resistance to stripe rust and leaf rust, respectively. Four of these QTL on chromosomes 1BL, 2AS, 2DS, and 7BL conferred resistance to both stripe rust and leaf rust. The QTL on 1BL, 2AS, and 7BL were identified as Lr46/Yr29, Lr37/Yr17, and Lr68, respectively. QYr.hbau-2DS/QLr.hbau-2DS was detected at similar positions to previously reported loci. QYr.hbau-1DL, QYr.hbau-3AS, and QYr.hbau-3DL are likely to be new. Combined effects of QTL in the RIL population indicated RIL combining all QTL had the highest resistance level compared with those of lower numbers or no QTL. These QTL, with their closely linked SNP markers, are applicable for marker-assisted breeding and candidate gene discovery.

First Report of Colletotrichum cereale Causing Anthracnose on Avena nuda in China.

Naked oats (Avena nuda L.) is rich in protein, fat, vitamin, mineral elements and so on, and is one of the world's recognized cereal crops with the highest nutritional and healthcare value. In July 2019, leaf spot was detected on A. nuda in Zhangbei experimental station of Hebei Agricultural University. The incidence of disease is 10% to 20%. The symptoms were similar to anthracnose disease, the infected leaves had fusiform or nearly fusiform yellowish-brown spots, yellow halo around the spots. Numerous acervuli with black setae diagnostic of fungi in the genus Colletotrichum were present on necrotic lesions. To identify the pathogen, ten symptomatic leaves were collected, and only one disease spot was isolated from each leaf. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and surface disinfested with 75% alcohol for 30s, 0.1% corrosive sublimate for 1 min, rinsed three times in sterile water. Plant tissues were then transferred on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Two fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. The aerial mycelium was gray black. The conidia were colorless and transparent, falcate, slightly curved, tapered toward the tips, and produced in acervuli with brown setae. The length and width of 100 conidia were measured and size ranged from 1.86 to 3.84 × 8.62 to 29.81 µm. These morphological characteristics were consistent with the description of Colletotrichum cereale (Crouch et al. 2006). To further assess the identity of the species, the genomic DNA of two fungal isolates (LYM19-4 and LYM19-10) was extracted by a CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region as well as, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and the beta-tubulin 2 (Tub2) partial genes were amplified and sequenced with primers ITS4/5, GDF/GDR, ACT-512F/ACT-783R, and T1/Bt2b, respectively (Carbone et al. 1999; Templeton et al. 1992; O'Donnell et al. 1997; Glass et al. 1995). The sequences of the ITS-rDNA region (MW040121, MW040122), the GAPDH sequences (MW052554, MW052555), the ACT sequences (MW052556, MW052551) and the Tub2 sequences (MW052552, MW052553) of the two single-spore isolates were more than 99% identical to C. cereale isolate CGMCC3.15110 (JX625159, KC843517, KC843534 and JX625186). Maximum likelihood tree based on concatenated sequences of the four genes were constructed using MEGA7. The results showed the strains isolated from A. nuda were closely related to C. cereale, as supported by high bootstrap values. A pathogenicity test of the C. cereale isolates was performed on first unfolding leaves of A. nuda. Koch's postulates were carried out with isolates by spraying a conidial suspension of 106 conidia/mL on leaves of healthy A. nuda. Four replicated pots were inoculated at a time, 10 leaves each pot, while sterile distilled water was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Anthracnose symptoms developed on the inoculated plants 7 days post inoculation while all control plants remained healthy. Microscopic examination showed the surface of infected leaves had the same acervuli, setae, and conidia as the original isolate. The pathogenicity test was repeated three times. C. cereale was previously reported as the causal agent of anthracnose on feather reed grass in US (Crouch et al. 2009). To our knowledge, this is the first report of C. cereale as the causal agent of A. nuda anthracnose in China.

Race and Virulence Analysis of Puccinia triticina in China in 2014 and 2015.

Wheat leaf rust, caused by Puccinia triticina, is an important fungal disease of wheat in China. To study races of the pathogen in China, leaf rust samples were collected from 14 provinces in 2014 and 15 provinces in 2015. From the samples, 494 single-uredinial isolates were derived from the 2014 collection and 649 from the 2015 collection. These isolates were tested on 40 near-isogenic lines of Thatcher carrying single leaf rust resistance genes. From the isolates, 84 races were identified in 2014 and 65 races in 2015. Races THTT (22.1%), THTS (19.6%), THJT (8.7%), PHTT (4.9%), and PHJT (3.6%) were the most common races in 2014, and THTT (28.4%), THTS (12.8%), THJT (11.6%), THJS (9.9%), and PHTT (9.7%) were the most frequent in 2015. All of these races were avirulent to resistance genes Lr9 and Lr24. THTT and THTS, the most frequent races in both years, were widely distributed throughout the country. The frequencies of isolates with virulence to Lr1, Lr2a, Lr2c, Lr3, Lr16, Lr26, Lr11, Lr17, LrB, Lr10, Lr14a, Lr2b, Lr3bg, Lr14b, Lr32, Lr33, and Lr50 were over 80%, whereas the frequencies of virulence to Lr9, Lr19, Lr25, Lr28, Lr29, and Lr47 were less than 3.5%. In the present study, all isolates were avirulent to Lr24 and Lr38. The race analysis and individual virulence frequencies provide guidance to breeders in choosing leaf rust resistance genes for use in breeding programs.

Identification of Wheat Leaf Rust Resistance Genes in Chinese Wheat Cultivars and the Improved Germplasms.

Leaf rust is an important wheat disease that is a significant hindrance for wheat production in most areas of the world. Breeding resistant cultivars can effectively and economically control the disease. In the present study, a wheat collection consisting of 100 cultivars from China and 18 improved germplasms from global landrace donors together with 36 known single Lr gene lines were tested with 20 strains of Puccinia triticina Eriks. in the seedling stage to postulate the Lr gene in the cultivars and germplasms. In addition, 12 diagnostic molecular markers specific to 10 Lr genes were used to detect the presence of the Lr genes in the wheat collection. Resistance to leaf rust of these cultivars at the adult plant stage was tested in fields under natural infection during the 2016 to 2018 cropping seasons in Baoding, Hebei Province. The gene postulation combined with molecular marker detection showed that six Lr genes (Lr1, Lr26, Lr33, Lr34, Lr45, and Lr46) were identified in 44 wheat accessions, including 37 cultivars and seven improved germplasms. Among the 44 wheat accessions postulated with Lr genes, Lr1 was present in four accessions, Lr26 in 12 accessions, Lr33 in two accessions, Lr34 in 14 accessions, Lr45 in three accessions, and Lr46 in 16 accessions. In the collection of 118 cultivars/germplasms, 34 wheat lines displayed adult-plant resistance carrying Lr34, Lr46, and/or underdetermined genes. Therefore, a high level of leaf rust resistance can be achieved through the combination of all-stage resistance and adult-plant resistance genes together in wheat cultivars.

Identification of Leaf Rust Resistance Genes in Bread Wheat Cultivars from Ethiopia.

Wheat leaf rust, caused by Puccinia triticina (Pt), is a widespread disease of bread wheat worldwide. In the present study, 50 wheat cultivars from Ethiopia and 34 differential lines, mostly near-isogenic lines (NILs) in the background of Thatcher with known resistance genes to leaf rust (Lr), were tested with 14 Pt races in the greenhouse to postulate Lr genes at the seedling stage. Field experiments were also conducted to identify adult plant responses to leaf rust in Baoding in the 2017-2018 and 2018-2019 growing seasons and in Zhoukou in the 2018-2019 growing season. Thirteen Lr genes (Lr1, Lr18, Lr3ka, Lr15, Lr26, Lr20, Lr14a, Lr30, Lr2a, Lr11, Lr34, Lr46, and Lr68) either singly or in combination were found in 39 cultivars. Known Lr genes were not present in the remaining 11 cultivars. Lr1 and Lr46, each in 13 cultivars, and Lr34 in 12 cultivars were the most commonly identified resistance genes. Less frequently identified genes included Lr26 (five cultivars); Lr30 and Lr18 (each present in four cultivars); Lr15, Lr3ka, and Lr2a (each identified in three cultivars); and Lr68 (two cultivars). Evidence for the existence of Lr11, Lr20, and Lr14a (each in one cultivar) was also obtained. Twenty-one cultivars were found to have slow rusting resistance to leaf rust in the field tests. The results should be valuable for cultivar selection with combinations of effective Lr genes and used in breeding new cultivars with improved resistance to leaf rust in Ethiopia and China.

First Report of Alternaria alternata Causing Leaf Spot on Avena nuda in Zhangbei, China.

Naked oats (Avena nuda L.) is an independent species of Avena, which can be used as both food and forage for rich nutritional value. In August 2019, leaf spot was observed at a naked oats planting base in Zhangbei County, Zhangjiakou City, Hebei Province. The incidence of disease was 40% to 50%. The symptoms of the lesions were chlorosis and gradually developing light brown spots with light yellow halos. The spots were irregular, enlarged and even coalesced to form large areas of necrosis on leaves. To identify the pathogen, twenty symptomatic leaves were collected, and one disease spot was isolated from each samples. Small square leaf pieces (3 to 5 mm) were excised from the junction of diseased and healthy tissues with a sterile scalpel and were sterilized with 75% alcohol for 30s, 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water, then transferred cultured on potato dextrose agar (PDA) at 25°C for 7 days. Four fungal isolates were obtained and purified by single-spore isolation method. All fungi have the same morphology and no other fungi were isolated. Colonies of the isolates had round margins, and thick fluffy aerial mycelia with brown coloration after 7 days on PDA. Conidiophores were brown, straight or flexuous, septate, single or in clusters. Conidia were obclavate or oval, dark brown, and size ranging from 4.61 to 15.68 × 6.61 to 35.49µm (n=100), with longitudinal and transverse septa varying from 1 to 3 and 1 to 7, respectively. The transverse median septum of the central section was especially thick. On the basis of morphological characteristics, the isolates were identified as Alternaria spp. (Simmons 2007). To further assess the identity of the species, the genomic DNA of pathogenic isolate (YM3) was extracted by CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase II second largest subunit (RPB2), and the plasma membrane ATPase genes were amplified and sequenced with primers ITS1/4, gpd1/2, RPB2-6F/7cR and ATPDF1/ATPDR1 respectively (Nishikawa and Nakashima 2015; Woudenberg et al. 2015). Sequences of ITS, GAPDH, RPB2 and ATPase (MN646900, MT233043, MT233042, MN640794) of the isolate was 99.82%, 99.68%, 100% and 99.51% similar to the fungus A. alternata (MK461082.1, MK451978, KP124770.1, MK804115). A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate YM3 clustered in the A. alternata clade with 100% bootstrap support. Therefore, the pathogen was identified as A. alternata based on the morphological characteristics and molecular identification. A pathogenicity test of the A. alternata isolates was performed by placing mycelial disks (5 mm) with conidia on the surface of the first unfolding leaves of naked oats. Each leaf was inoculated with three disks. The pathogenicity test was repeated four times, and 10 leaves were inoculated in each repetition, while sterile PDA was used as the control. All treated plants were placed in a moist chamber (25°C, 16-h light and 8-h dark period). Leaf spot symptoms developed on the inoculated plants about 10 days post inoculation while all control plants remained healthy. The similar isolates were re-isolated from the inoculated and infected leaves and identified as A. alternata by DNA sequencing, fulfilling Koch's postulates. It has been reported that A. alternata can cause leaf spots on A. Sativa(Chen et al. 2020). However, to our knowledge, this is the first report of A. alternata causing leaf spots on A. nuda in China. It can be concluded that A. alternata can cause leaf spot disease of oats (A. Sativa and A. nuda). The spots disease is worthy of our attention for its harm to the production of oats.

Race and Virulence Analysis of Puccinia triticina in China During 2011 to 2013.

Wheat leaf rust, caused by Puccinia triticina, is a common fungal disease of wheat in China. In order to identify races and determine the individual virulence of isolates in different wheat-growing regions in China, leaf rust samples collected from 18 provinces in 2011 to 2013 were tested on 37 Thatcher near-isogenic lines each carrying a different single leaf rust resistance gene. A total of 158 races were identified. Races THTT (19.5%), THTS (16.9%), PHTT (7.7%), THJS (5.0%), THJT (4.2%), and PHTS (4.0%) were the most predominant races in 2011 to 2013. All of these races were avirulent to resistance genes Lr9 and Lr24. The two most frequent races, THTT and THTS, were widely distributed. The frequencies of the isolates with virulence to Lr1, Lr2c, Lr3, Lr16, Lr26, Lr17, LrB, Lr10, Lr14a, Lr3bg, Lr14b, Lr33, Lr37, and Lr50 exceeded 90%. Frequencies of virulence to Lr2a, Lr3ka, Lr11, Lr30, Lr2b, and Lr32 exceeded 70% but were less than 90%. Frequencies of virulence to Lr18, Lr21, Lr15, Lr23, Lr33+34, Lr36, Lr39, and Lr44 were below 70%, whereas the frequency of virulence to Lr25 was less than 1%. All isolates were avirulent to Lr9, Lr19, Lr24, Lr28, Lr42, Lr29, Lr38, and Lr47. The identified races and individual virulence frequencies provide a basis for selection of effective leaf rust resistance genes for use in breeding programs and can also provide information for the study of race evolution of P. triticina.

Treatment with Subcritical Water-Hydrolyzed Citrus Pectin Ameliorated Cyclophosphamide-Induced Immunosuppression and Modulated Gut Microbiota Composition in ICR Mice.

Subcritical water can effectively hydrolyze pectin into smaller molecules while still maintaining its functional regions. Pectic heteropolysaccharide can mediate immune regulation; however, the possible effects of subcritical water-hydrolyzed citrus pectin (SCP) on the immune response remain unclear. Therefore, the effects of SCP on immunomodulatory functions and intestinal microbial dysbiosis were investigated using a cyclophosphamide-induced immunosuppressed mouse model. In this research, immunosuppressed ICR mice were administrated with SCP at dosages of 300/600/1200 mg/kg.bw by oral gavage, and body weight, immune organ indexes, cytokines, and gut microbiota were determined. The results showed that subcritical water treatment decreased the molecular mass and increased the content of galacturonic acid in citrus pectin hydrolysates. Meanwhile, the treatment with SCP improved immunoregulatory functional properties and bioactivities over the original citrus pectin. For example, SCP protected immune organs (accelerated recovery of immune organ indexes) and significantly enhanced the expression of immune-related cytokines (IL-2, IL-6, IFN-γ, and TNF-α). The results of the 16S rDNA sequencing analysis on an IlluminaMiSeq platform showed that SCP normalized Cy-induced gut dysbiosis. SCP ameliorated Cy-dependent changes in the relative abundance of several taxa, shifting the balance back to normal status (e.g., SCP increased beneficial Muribaculaceae, Ruminococcaceae, Bacteroidaceae, and Prevotellaceae while decreasing pathogenic Brevundimonas and Streptococcus). The results of this study suggest an innovative application of citrus pectin as an immunomodulator.