First report of Lasiodiplodia pseudotheobromae causing soft rot of plum in China.

Plum (Prunus salicina) is one of the most important fruit tree species worldwide (Valderrama-Soto et al. 2021). In June 2023, the postharvest soft rot symptoms were observed on plum fruits in several fruit markets of Guiyang city, Guizhou province, China. The disease incidence in these markets ranged from 20 to 25% with 70% disease severity. Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. In severe conditions, whole fruits become rotted and were covered with white fungal mycelia. Small sections (5 × 3 mm) from 6 diseased plum fruits were surface sterilized by using 75% ethanol for 30 s followed by 0.1% mercuric chloride solution for 5 min, rinsed three times with ddH2O, and then transferred onto potato dextrose agar (PDA) and incubated at 25 ± 2°C for three days. Three pure cultures (GUCC23-0001 to GUCC23-0003) were obtained by transferring a single hyphal tip to new PDA plates. Colonies of these isolates were grayish-white initially, gradually turning to whitish brown with fluffy aerial mycelia and uneven edges and finally turned to a dark gray colony after five days of inoculation. The pseudoparaphyses were hyaline, cylindrical, aseptate, and rounded at apex. Conidia were ellipsoidal, hyaline, unicellular, and 24.2 to 28.6 × 12.3 to 15.5 µm in size (n = 30) (Fig. S1), which were similar to the morphology of Lasiodiplodia pseudotheobromae (Alves et al. 2008). Furthermore, fungal DNA was extracted from fresh mycelia of PDA after seven days by using fungus genomic DNA extraction kit (Biomiga, USA). Partial DNA sequences from four loci including internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and polymerase II second largest subunit (rpb2) were amplified with ITS1 and ITS4 (White et al. 1990), EF1-688F and EF1-1251R (Alves et al. 2008), Bt2a and Bt2b (Glass and Donaldson 1995), and RPB2-LasF and RPB2-LasR, respectively (Cruywagen et al. 2017). GenBank accession numbers are OR361680, OR361681, OR361682 for ITS, OR423394, OR423395, OR423396 for tef1, OR423397, OR423398, OR423399 for tub2, and OR423391, OR423392, OR423393 for rpb2, and gene sequencing showed 99.6 to 100% identity with ex-type strain of L. pseudotheobromae (CBS 116459). Phylogenetic analysis also placed our isolates in a highly supported clade with the reference isolate of L. pseudotheobromae (Fig. S2). Another experiment was designed to confirm the pathogenicity test for additional confirmation. Five mm mycelial plugs of L. pseudotheobromae from a three day old culture on PDA were placed on five surface-sterilized and non-wounded plum fruits for 12 hours and incubated at 25°C ± 2°C for four days. Sterilized fungus free PDA plugs were used as a negative control. Mycelial plugs were removed after 12 hours following which whole fruits were incubated in plastic boxes at 25°C ± 2°C. The experiment was repeated twice. The pathogenicity was evaluated under control conditions in laboratory (relative humidity, 70 ± 5% and temperature 25 ± 5˚C). Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. These symptoms and signs were similar to the initially observed symptoms on plums in the markets. No disease symptoms were observed on the control fruits. The re-isolated fungus obtained from inoculated plum fruits was very similar to those isolated from diseased samples in morphology, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of L. pseudotheobromae causing postharvest fruit rot of plum in China. In 2022, the total planting area of plum was 1946.5 thousand hectares, which produces approximately 6626300 tons of plum (Food and Agriculture Organization of the United Nations, 2022). Based on the disease incidence and severity reported in the current study, soft rot of plum may be responsible for nearly 35% of yield losses under severe. Therefore, our study laid a theoretical foundation for the prevention and control of this post-harvest disease of plum.

First report of wheat dwarf bunt caused by Tilletia controversa Kühn in Pakistan.

Wheat (Triticum aestivum L.) is critical to food security worldwide. Wheat dwarf bunt is caused by Tilletia controversa Kühn and can cause 70-80% losses under severe condition (Trione et al. 1989; Xu et al., 2021). In May 2022, we observed dwarf bunt disease in six fields grown with spring cultivar (Glaxy-13) in District Swat, KPK-Pakistan. Infected plants had mottling and flecking on leaves, a greater number of tillers and were smaller than healthy plants. Diseased wheat head spikes were larger, wider and thicker, had bunted kernels (sori) filled with brown-black teliospores and a strong odor like that of rotten fish. Individual fields showed 10% infected plants while no dwarf bunt was recorded in nearby fields. About 150 heads exhibiting bunted kernels were collected among the six fields. Kernels were surface sterilized with 30% NaClO for 5 min after crushing by a centrifuge machine and washed with ddH20 three times. The teliospore suspension (1×106 spores/mL) was spread on 2% soil agar plates in a growth chamber (MLR 352 H, Panasonic, USA) and incubated at 5°C with 60% relative humidity for 60 days to test for T. controversa germination or at 16°C and 60% relative humidity for 15 days (MLR 352 H, Panasonic, USA) to test for T. caries and T. laevis germination. Teliospores germinated only on plates kept at 5°C. Teliospores were morphologically identified as a T. controversa from the infected samples. They ranged in size from 15.0 to 20.5 µm diam. and the walls had deep reticulations surrounded by a transparent sheath, differing from T. laevis which has smooth teliospores and T. caries which has no sheath and reticulations on the surface (Mathre 1996). To further confirm Tilletia spp. identification, genomic DNA of our two isolates (gmd123 and gmd1234) was obtained using an extraction kit (TransGen, Beijing, China). The internal transcribed spacer (ITS) region was amplified by using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession no. OR366448 and OR366450 provided additional evidence the isolates belong to the complex of species that includes the three bunt species causing diseases on wheat, with 100% matches to verified sequences for T. controversa (eg. EU257561) but also to T. laevis and T. caries. Based on disease symptoms, teliospore morphology, germination at 5°C but not at 16°C, the bunt fungus was identified as T. controversa. To fulfill Koch's postulates, 10 mL (106 spores/mL) of germinated teliospores were injected into rhizosphere soil of Galaxy-13 cultivar at 2 leaves unfolded growth stage (Zadoks 12) and 2 mL (106 spores/mL) were injected into heads of same plants at growth stages Zadoks 61-65 with a syringe. Plants injected with sterile ddH2O were used as a control. Inoculated plants were grown in a growth chamber at 8°C with 50% humidity and 24 h light. After one month at the ripening stage, the bunted kernels of the inoculated plants were filled with black teliospores releasing a fishy smell, and the control plants did not have bunted kernels. Under an optical microscope, teliospores from the inoculated plants had reticulation surface and were measured 15 to 20.5 µm in diameter, similar to the teliospores of bunt heads from the fields. To the best of our knowledge, this is the first report of T. controversa causing dwarf bunt in district Swat, KPK-Pakistan. Because the pathogen is seedborne and soilborne, the disease may become a high risk to wheat production in Pakistan. Therefore, detection of this pathogen is very important to control the disease on time.

Ceratocystis fimbriata Causing Postharvest Fruit Rot of Pear in Punjab, Pakistan.

Pear (Pyrus communis) is an important deciduous fruit cultivated on a worldwide scale including Pakistan. During August 2021, a postharvest fruit rot disease was observed on several pears at various farmers market in Okara- a district of Punjab Province, Pakistan. The incidence of the disease varied from 7 to 20% with 35% disease severity. Necrotic spots (10 to 20 mm diameter) were first observed on the infected pear fruit. The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium. The isolates were obtained from the symptomatic tissues (n = 18) incubated on carrot discs that had been surface sterilized in 100-ppm streptomycin solution. After consistent sporulation of a fungus on the carrot discs, the ascospore masses formed at the tip of perithecia were transferred to malt extract agar (MEA). Primary conidia were cylindrical and hyaline (7 to 11 × 4 to 7 µm) and secondary conidia were hyaline and barrel-shaped (7 to 12 × 5 to 8 µm). Endoconidiophores with primary conidia were (12 to 27 × 2.6 to 5.5 µm). Perithecia produced on carrot discs were dark brown to black, and the base was 157 to 278 µm in diameter. Ascomatal necks were 512 to 656 µm long, dark brown to black, lighter in color at apices, tapering from base (23 to 45 µm diameter) to apex (13 to 24 µm diameter). Ostiolar hyphae were 41 to 79 µm long. Ascospores were hyaline, hat shaped, 3 to 4 µm long, and accumulated in a sticky matrix at the tips of perithecial necks. Mycelium was initially hyaline but became dark greenish brown after 7 days. Dark brown, thick-walled aleuroconidia (13 to 19.5 × 9 to 14 µm) appeared on culture plates after 2 months. Based on morphological characteristics, the fungus was identified as Ceratocystis fimbriata (Engelbrecht, 2005; Suwandi et al. 2021). To further confirm species identification, genomic DNA of two representative isolates (UO-05 and UO-06) was obtained using an extraction kit. The internal transcribed spacer (ITS) region was amplified using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession nos. OR185451 and OR185456 indicated 99 to 100% identity with several C. fimbriata including type species (MH856050.1; KC493160.1; MT560374.1). Pathogenicity tests were conducted by inoculating nine disease-free pear (cv. Concord) fruit after disinfesting in 75% ethanol. A prepared spore suspension (1.0 × 106 spores/ml) was dropped on the wounds (a depth of 1 mm diameter) on the pear surface, which were made by a sterilized needle. 10 µl of a prepared spore suspension was dropped onto nine pears. Sterile water (10 µl) was dropped on the wounded sites of nine pear fruits as negative controls and all fruits were incubated in a growth chamber at 30/26°C (day/night, 90% relative humidity). Symptoms similar to those on the naturally infected fruits began after 4 to 5 days of inoculation, while controls remained healthy. The fungal isolates recovered from inoculated pears were morphologically identical to the C. fimbriata isolates originally recovered from symptomatic fruits fulfilling Koch's postulates. The pathogen has been reported to cause postharvest fruit rot of passion fruit and cucumber (Firmino et al. 2016; Li et al. 2019). To our knowledge, this is the first report of C. fimbriata causing fruit rot of pear in Punjab Province. The detection of this disease will help pear growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

Characterization of histological changes at the tillering stage (Z21) in resistant and susceptible wheat plants infected by Tilletia controversa Kühn.

BACKGROUND: Dwarf bunt, which is caused by Tilletia controversa Kühn, is a soilborne and seedborne disease that occurs worldwide and can lead to 70% or even total losses of wheat crops. However, very little information is available about the histological changes that occur in dwarf bunt-resistant and dwarf bunt-susceptible wheat plants at the tillering stage (Z21). In this study, we used scanning electron microscopy and transmission electron microscopy to characterize the histological changes at this stage in resistant and susceptible wheat cultivars infected by T. controversa. RESULTS: Using scanning electron microscopy, the root, stem, and leaf structures of resistant and susceptible cultivars were examined after T. controversa infection. The root epidermal and vascular bundles were more severely damaged in the susceptible T. controversa-infected plants than in the resistant plants. The stem cell and longitudinal sections were much more extensively affected in susceptible plants than in resistant plants after pathogen infection. However, slightly deformed mesophyll cells were observed in the leaves of susceptible plants. With transmission electron microscopy, we found that the cortical bundle cells and the cell contents and nuclei in the roots were more severely affected in the susceptible plants than in the resistant plants; in the stems and leaves, the nuclei, chloroplasts, and mesophyll cells changed significantly in the susceptible plants after fungal infection. Moreover, we found that infected susceptible and resistant plants were affected much more severely at the tillering stage (Z21) than at the seedling growth stage (Z13). CONCLUSION: Histological changes in the wheat roots, stems and leaves were much more severe in T. controversa-infected susceptible plants than in infected resistant plants at the tillering stage (Z21).

Intelligent reprogramming of wheat for enhancement of fungal and nematode disease resistance using advanced molecular techniques.

Wheat (Triticum aestivum L.) diseases are major factors responsible for substantial yield losses worldwide, which affect global food security. For a long time, plant breeders have been struggling to improve wheat resistance against major diseases by selection and conventional breeding techniques. Therefore, this review was conducted to shed light on various gaps in the available literature and to reveal the most promising criteria for disease resistance in wheat. However, novel techniques for molecular breeding in the past few decades have been very fruitful for developing broad-spectrum disease resistance and other important traits in wheat. Many types of molecular markers such as SCAR, RAPD, SSR, SSLP, RFLP, SNP, and DArT, etc., have been reported for resistance against wheat pathogens. This article summarizes various insightful molecular markers involved in wheat improvement for resistance to major diseases through diverse breeding programs. Moreover, this review highlights the applications of marker assisted selection (MAS), quantitative trait loci (QTL), genome wide association studies (GWAS) and the CRISPR/Cas-9 system for developing disease resistance against most important wheat diseases. We also reviewed all reported mapped QTLs for bunts, rusts, smuts, and nematode diseases of wheat. Furthermore, we have also proposed how the CRISPR/Cas-9 system and GWAS can assist breeders in the future for the genetic improvement of wheat. If these molecular approaches are used successfully in the future, they can be a significant step toward expanding food production in wheat crops.

Transcriptomics Analysis of Wheat Tassel Response to Tilletia laevis Kühn, Which Causes Common Bunt of Wheat.

Tilletia laevis Kühn [synonym T. foetida (Wallr.) Liro] can lead to a wheat common bunt, which is one of the most serious diseases affecting kernels, a serious reduction in grain yield, and losses can reach up to 80% in favorable environments. To understand how wheat tassels respond to T. laevis, based on an RNA-Seq technology, we analyzed a host transcript accumulation on healthy wheat tassels and on tassels infected by the pathogen. Our results showed that 7,767 out of 15,658 genes were upregulated and 7,891 out of 15,658 genes were downregulated in wheat tassels. Subsequent gene ontology (GO) showed that differentially expressed genes (DEGs) are predominantly involved in biological processes, cellular components, and molecular functions. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that 20 pathways were expressed significantly during the infection of wheat with T. laevis, while biosynthesis of amino acids, carbon metabolism, and starch and sucrose metabolism pathways were more highly expressed. Our findings also demonstrated that genes involved in defense mechanisms and myeloblastosis (MYB) transcription factor families were mostly upregulated, and the RNA-seq results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report on transcriptomics analysis of wheat tassels in response to T. laevis, which will contribute to understanding the interaction of T. laevis and wheat, and may provide higher efficiency control strategies, including developing new methods to increase the resistance of wheat crops to T. laevis-caused wheat common bunt.

ITRAQ-Based Proteomic Analysis of Wheat (Triticum aestivum) Spikes in Response to Tilletia controversa Kühn and Tilletia foetida Kühn Infection, Causal Organisms of Dwarf Bunt and Common Bunt of Wheat.

Dwarf bunt and common bunt diseases of wheat are caused by Tilletia controversa Kühn and Tilletia foetida Kühn, respectively, and losses caused by these diseases can reach 70-80% in favourable conditions. T. controversa and T. foetida are fungal pathogens belonging to the Exobasidiomycetes within the basidiomycetous smut fungi (Ustilaginomycotina). In order to illuminate the proteomics differences of wheat spikes after the infection of T. controversa and T. foetida, the isobaric tags for relative and absolute quantification (iTRAQ) technique was used for better clarification. A total of 4553 proteins were differentially detected after T. controversa infection; 4100 were upregulated, and 453 were downregulated. After T. foetida infection, 804 differentially expressed proteins were detected; 447 were upregulated and 357 were downregulated. In-depth data analysis revealed that 44, 50 and 82 proteins after T. controversa and 9, 6 and 16 proteins after T. foetida were differentially expressed, which are antioxidant, plant-pathogen interaction and glutathione proteins, respectively, and 9 proteins showed results consistent with PRM. The top 20 KEGG enrichment pathways were identified after pathogen infection. On the basis of gene ontology, the upregulated proteins were linked with metabolic process, catalytic activity, transferase activity, photosynthetic membrane, extracellular region and oxidoreductase activity. The results expanded our understanding of the proteome in wheat spikes in response to T. controversa and T. foetida infection and provide a basis for further investigation for improving the defense mechanism of the wheat crops.

Metabolomic Analysis of Wheat Grains after Tilletia laevis Kühn Infection by Using Ultrahigh-Performance Liquid Chromatography-Q-Exactive Mass Spectrometry.

Tilletia laevis causes common bunt disease in wheat, with severe losses of production yield and seed quality. Metabolomics studies provide detailed information about the biochemical changes at the cell and tissue level of the plants. Ultrahigh-performance liquid chromatography-Q-exactive mass spectrometry (UPLC-QE-MS) was used to examine the changes in wheat grains after T. laevis infection. PCA analysis suggested that T. laevis-infected and non-infected samples were scattered separately during the interaction. In total, 224 organic acids and their derivatives, 170 organoheterocyclic compounds, 128 lipids and lipid-like molecules, 85 organic nitrogen compounds, 64 benzenoids, 31 phenylpropanoids and polyketides, 21 nucleosides, nucleotides, their analogues, and 10 alkaloids and derivatives were altered in hyphal-infected grains. According to The Kyoto Encyclopedia of Genes and genomes analysis, the protein digestion and absorption, biosynthesis of amino acids, arginine and proline metabolism, vitamin digestion and absorption, and glycine, serine, and threonine metabolism pathways were activated in wheat crops after T. laevis infection.

Characterization of Rhizosphere Microbial Communities for Disease Incidence and Optimized Concentration of Difenoconazole Fungicide for Controlling of Wheat Dwarf Bunt.

Rhizosphere soil microorganisms have great agricultural importance. To explore the relationship between rhizosphere microorganisms and the disease incidence, and to optimize the concentration of difenoconazole fungicide for the control of wheat dwarf bunt, caused by Tilletia controversa Kühn, the rhizosphere microorganisms were characterized based on sequencing methods. We found that the disease incidence correlated with the relative abundance of some microbial communities, such as Acidobacteria, Nocardioides, Roseiflexaceae, Pyrinomonadaceae, and Gemmatimonadaceae. Actinobacteria showed significant differences in the infected soils when compared to the control soils, and the relative abundance of Acidobacteria, Pyrinomonadaceae, Gemmatimonadaceae, and Saccharimonadales populations was distinctly higher in the T. controversa-inoculated group than in the control group. The members of Dehalococcoidia, Nitrosomonadaceae, and Thermomicrobiales were found only in T. controversa-inoculated soils, and these taxa may have potential effects against the pathogen and contribute to disease control of wheat dwarf bunt. In addition, for T. controversa-infected plants, the soil treated with difenoconazole showed a high relative abundance of Proteobacteria, Actinobacteria, Ascomycota, Basidiomycota, Mortierellomycota, and Olpidiomycota based on the heatmap analysis and ANOVA. Our findings suggest that the optimized concentration of fungicide (5% recommended difenoconazole) exhibits better control efficiency and constant diversity in the rhizosphere soil.

Development of real-time PCR and droplet digital PCR based marker for the detection of Tilletia caries inciting common bunt of wheat.

This is the first study reporting droplet digital PCR and quantitative real time PCR for detection of Tilletia caries (syn. T. tritici), which causes common bunt of wheat and leads to yield losses of 80% in many wheat growing areas worldwide. To establish an accurate, rapid and quantifiable detection method, we tested 100 inter simple sequence repeats (ISSR) primers and obtained a species-specific fragment (515 bp) generated by ISSR 827. Then, a specific 266 bp band for the sequence characterized amplified region (SCAR) marker was produced from T. caries. The detection limit reached 50 pg/µL. Based on the SCAR marker, we further developed a higher sensitivity of quantitative real time-polymerase chain reaction (qRT-PCR) with a detection limit of 2.4 fg/µL, and droplet digital PCR (ddPCR) with a detection limit of 0.24 fg/µL. Both methods greatly improved the detection sensitivity of T. caries, which will be contribute a lot for quickly and accurately detection of T. caries, which causes wheat common bunt.

Wheat Varietal Response to Tilletia controversa J. G. Kühn Using qRT-PCR and Laser Confocal Microscopy.

Tilletia controversa J. G. Kühn is a causal organism of dwarf bunt in wheat. Understanding the interaction of wheat and T. controversa is of practical and scientific importance for disease control. In this study, the relative expression of TaLHY and TaPR-4 and TaPR-5 genes was higher in a resistant (Yinong 18) and moderately resistant (Pin 9928) cultivars rather than susceptible (Dongxuan 3) cultivar at 72 h post inoculation (hpi) with T. controversa. Similarly, the expression of defensin, TaPR-2 and TaPR-10 genes was observed higher in resistant and moderately resistant cultivars after exogenous application of phytohormones, including methyl jasmonate, salicylic acid, and abscisic acid. Laser confocal microscopy was used to track the fungal hyphae in the roots, leaves, and tapetum cells, which of susceptible cultivar were infected harshly by T. controversa than moderately resistant and resistant cultivars. There were no fungal hyphae in tapetum cells in susceptible cultivar after methyl jasmonate, salicylic acid and abscisic acid treatments. Moreover, after T. controversa infection, the pollen germination was of 80.06, 58.73, and 0.67% in resistant, moderately resistant and susceptible cultivars, respectively. The above results suggested that the use using of resistant cultivar is a good option against the dwarf bunt disease.

Metabolomics analysis of grains of wheat infected and noninfected with Tilletia controversa Kühn.

Dwarf bunt caused by the pathogen Tilletia controversa Kühn is one of the most serious quarantine diseases of winter wheat. Metabolomics studies provide detailed information about the biochemical changes at the cell and tissue levels of plants. In the present study, a liquid chromatography/mass spectrometry (LC/MS) metabolomics approach was used to investigate the changes in the grain metabolomics of infected and noninfected with T. controversa samples. PCA suggested that T. controversa-infected and noninfected samples were separated during the interaction. LC/MS analysis showed that 62 different metabolites were recorded in the grains, among which a total of 34 metabolites were upregulated and 28 metabolites were downregulated. Prostaglandins (PGs) and 9-hydroxyoctadecadienoic acids (9-HODEs) are fungal toxin-related substances, and their expression significantly increased in T. controversa-infected grains. Additionally, the concentrations of cucurbic acid and octadecatrienoic acid changed significantly after pathogen infection, which play a large role in plant defense. The eight different metabolic pathways activated during T. controversa and wheat plant interactions included phenylalanine metabolism, isoquinoline alkaloid biosynthesis, starch and sucrose metabolism, tyrosine metabolism, sphingolipid metabolism, arginine and proline metabolism, alanine, aspartate, and glutamate metabolism, and tryptophan metabolism. In conclusion, we found differences in the metabolic profiles of wheat grains after T. controversa infection. To our knowledge, this is the first study to evaluate the metabolites in wheat grains after T. controversa infection.

Methyljasmonate and salicylic acid contribute to the control of Tilletia controversa Kühn, causal agent of wheat dwarf bunt.

Tilletia controversa Kühn (TCK) is the causal agent of dwarf bunt of wheat, a destructive disease in wheat-growing regions of the world. The role of Meja, SA and Meja + SA were characterized for their control of TCK into roots, coleoptiles and anthers. The response of the defence genes PR-10a, Catalase, COI1-1, COII-2 and HRin1 was upregulated by Meja, SA and Meja + SA treatments, but Meja induced high level of expression compared to SA and Meja + SA at 1, 2, and 3 weeks in roots and coleoptiles, respectively. The severity of TCK effects in roots was greater at 1 week, but it decreased at 2 weeks in all treatments. We also investigated TCK hyphae proliferation into coleoptiles at 3 weeks and into anthers to determine whether hyphae move from the roots to the upper parts of the plants. The results showed that no hyphae were present in the coleoptiles and anthers of Meja-, SA- and Meja + SA-treated plants, while the hyphae were located on epidermal and sub-epidermal cells of anthers. In addition, the severity of hyphae increased with the passage of time as anthers matured. Bunted seeds were observed in the non-treated inoculated plants, while no disease symptoms were observed in the resistance of inducer treatments and control plants. Plant height was reduced after TCK infection compared to that of the treated inoculated and non-inoculated treatments. Together, these results suggested that Meja and SA display a distinct role in activation of defence genes in the roots and coleoptiles and that they eliminate the fungal pathogen movement to upper parts of the plants with the passage of time as the anthers mature.

Characterization of the wheat cultivars against Tilletia controversa Kühn, causal agent of wheat dwarf bunt.

Wheat is one of the most important staple crops. Tilletia controversa Kühn is the causal agent of wheat dwarf bunt. In this study, a resistant wheat cultivar displayed significantly higher expression of pathogenesis-related genes than a susceptible cultivar at 7 days post inoculation (DPI) with T. controversa. Similarly, the expression was high in the resistant cultivar after exogenous application of phytohormones, including salicylic acid. The expression of pathogenesis-related genes, especially chitinase 4, was high in the resistant cultivar, while LPT-1 was down regulated after T. controversa infection. Callose deposition was greater in the resistant cultivar than in the susceptible cultivar at 10 DPI. Confocal microscopy was used to track the fungal hyphae in both cultivars in anther and ovary cells. The anthers and ovaries of the susceptible cultivar were infected by T. controversa at 7 and 15 DPI. There were no fungal hyphae in anther and ovary cells in the resistant cultivar until 10 and 23 DPI, respectively. Moreover, anther length and width were negatively influenced by T. controversa at 16 DPI. The plant height was also affected by fungal infection. Ultimately, resistance to T. controversa was achieved in cultivars via the regulation of the expression of defense-related and pathogenesis-related genes.

Development of the Droplet Digital PCR to Detect the Teliospores of Tilletia controversa Kühn in the Soil With Greatly Enhanced Sensitivity.

BACKGROUND AND AIMS: The dwarf bunt disease of wheat is caused by Tilletia controversa Kühn. This pathogen is primarily involved in the stunted growth of wheat and affects seed quality. Many countries in the world have therefore imposed quarantine bans to prevent the spread of T. controversa. Morphological observations are the main method of detecting teliospores in soil. However, this is a lengthy and laborious process; this method is thus unable to quickly meet the demand for detection of teliospores in the soil. METHODS: We compared PCR, real-time PCR and droplet digital PCR (ddPCR) for the qualitative and quantitative measurement of the teliospores of T. controversa in soil. RESULTS: We suggest the use of ddPCR for detection of the soil samples, which was demonstrated to have the most sensitive detection at 2.1 copies/µL. In contract, SYBR Green I real-time PCR could detect 7.97 copies/µL of T. controversa in soil, and this sensitivity was 100 times more sensitive than that of simple PCR. CONCLUSION: This study was the first report using ddPCR techniques to detect T. controversa teliospores in soil with greatly enhanced sensitivity.