Disease investigation, collection of diseased buds, isolation and purification of pathogens were conducted to clarify the species of Diaporthe causing pear bud witherings in Guizhou. In total, 94 Diaporthe isolates were obtained based on their colony characteristics combined with ITS sequence identification, of which 40 representative isolates were chosen for carrying out multi-gene (ITS, TEF, CAL, HIS and TUB) phylogenetic analysis and morphological observation. Eight species of Diaporthe were identified, including D. biguttulata, D. eres, D. ellipsoidea, D. fusicola, D. hongkongensis, D. sennae, D. sojae and D. unshiuensis. To our knowledge, D. ellipsoidea, D. biguttulata and D. sennae were first reported to infect pears. Inoculating in vitro branch of “Yuluxiang” pear showed that the eight species of Diaporthe could produce symptoms the same as those in the field, indicating that they were the pathogens causing pear bud witherings. D. eres with the highest isolation rate was dominant and had stronger pathogenicity. This is the first report of Diaporthe species causing pear bud witherings, providing useful data for practicable disease control of pear in Guizhou Province.
In recent years, guava wilt, with the typical symptoms of withered branch, necrotic trunk and whole plant dead, has been seriously occurred in Nansha district of Guangzhou. To clarify the cause of the disease, infectivity investigation, specimen collection, pathogen isolation, and pathogenicity test were carried out. Comparative morphology observation and phylogenetic analysis based on multiple gene sequences revealed that the pathogen of guava wilt occurred in Nansha district of Guangzhou was Nalanthamala psidii. Pathogenicity tests on different parts of the host showed that the pathogen not only could invade the host from injured branches, but also from the roots, causing slow-growth of the plant, withered branch and eventual whole plant death. The pathogen could also infect the fruits and cause fruit necrosis. This study confirmed that the guava wilt in Nansha district of Guangzhou was same with rapid death syndrome of guava reported in Taiwan Island of China, Malaysia and South Africa, which were caused by Na. psidii. This study provides a basis for further research of infection characteristics and efficient control of guava wilt.
Glomerella leaf spot caused by Colletotrichum fructicola is a severe disease on apple fruit and leaves in China. There are plus and minus strains with morphological and genetic differentiation in Colletotrichum fungi. However, the mechanisms underlying the differentiation between two types of strains remain poorly understood. The functions of CfAtg8 were analyzed based on gene deletion analysis with homologous recombination and PEG-mediated protoplast transformation. It was found that CfAtg8 significantly affected the colony color and aerial hypha density of the plus strain (1104-7), but it did not affect the minus strain (1104-6). CfAtg8 regulated the perithecial development. The plus mutant could not produce perithecia and the perithecia produced by minus mutant were more compacted and smaller in size. When Δ+CfAtg8/1104-6 was co-cultured, mating line formation failed, while a strong mating line developed at the contact zone between Δ-CfAtg8/1104-7. Additionally, CfAtg8 is involved in conidial germination, appressorium formation and pathogenicity in plus and minus strains. Our results reveal that CfAtg8 is an important gene regulating genetic differentiation of plus-minus strains, morphological development and pathogenicity in Colletotrichum fructicola.
Colletotrichum fructicola is a devastating fungal phytopathogen that infects and damages many plants. In certain filamentous fungi, deleting ku70 or ku80, key genes of the nonhomologous end joining (NHEJ) pathway, can significantly boost homologous recombination and thereby increase target gene replacement frequency. In this study, Cfku70 and Cfku80 genes were identified from the C. fructicola genome, and the impacts of their inactivation on fungal biology and gene deletion efficiency were characterized. Deleting Cfku70 or Cfku80 did not affect fungal colony morphology, vegetative growth, sporulation, conidial germination, appressorium development and pathogenicity; in the meanwhile, Cfku70 gene deletion considerably improved deletion efficiencies of three tested genes. Our results demonstrate the usefulness of ΔCfku70 as a chassis strain enabling high-efficient target gene knock-out, and lay an important foundation for identifying novel virulence factors through the large scale gene deletion strategy in C. fructicola.
Peroxisomes are single-membrane-bound organelles in eukaryotic cells involved in diverse metabolic processes. Pex13 and Pex14 are docking complex proteins located on the peroxisomal membrane, participating in the transport of matrix protein-receptor complex across the membrane. Currently, the biological functions of Pex13 and Pex14 in phytopathogenic fungi are poorly understood. In this study, the docking complex proteins Pex13 and Pex14 of the tangerine pathotype of Alternaria alternata were identified, and knockout mutants and complementation strain were constructed to explore their biological functions. The results showed that compared with wild-type and complementation strains, the vegetative growth, conidial formation, the germination rate of conidia, and the maintenance of antioxidant ability and resistance to cell-wall stress agents in ΔAaPex13 and ΔAaPex14 mutants were significantly decreased. The production of ACT toxin was reduced by 30% and 33%, respectively, and the pathogenicity on detached leaves was lost. In addition, deletion of AaPex13 and AaPex14 caused mis-localization of peroxisomal matrix proteins and impaired peroxisome biogenesis. These results prove that AaPex13 and AaPex14 play a critical role in mediating the development, peroxisome biogenesis, ACT toxin production and pathogenicity in the tangerine pathotype of A. alternata.
Glomerella leaf spot (GLS) caused by Colletotrichum fructicola is an important disease in apple production areas in China. During host infection, the fungus releases effector protein CfEC92 to assist pathogenesis. In this study, Mal d 1j protein was cloned from apple cDNA. Mal d 1j domain and amino acid sequence analysis showed that it was a member of the PR10 family. Overexpression of Mal d 1j significantly enhanced the resistance of apple to GLS, and silencing Mal d 1j attenuated the resistance effect. Overexpression of Mal d 1j enhanced tobacco resistance, and co-expression of CfEC92 and Mal d 1j attenuated Mal d 1j resistance to Phytophthora nicotianae. The interaction between CfEC92 and Mal d 1j was validated by yeast two-hybrid, BIFC and Co-IP analysis, and the interaction was co-localized in the cell membrane and nucleus, indicating that CfEC92 affected plant immunity by interacting with Mal d 1j. The results revealed that C. fructicola effector protein CfEC92 inhibited plant immunity and promoted the fungal infection through targeting Mal d 1j. These findings provide new insights for the prevention of GLS.
Apple moldy core caused by Trichothecium roseum is a severe disease. The infection route of T. roseum on Starkrimson Delicious cultivar is unclear. This research traced the pathogen colonization process of T. roseum on flower and fruit tissues of Starkrimson Delicious apple using a fluorescence labeling strain TR45. At the flowering period, the pathogen can colonize flower tissues, and cause necrosis and wilt symptom. In 10 to 15 days after flower fall, most infected flower residues accumulated within calyx tube. The pathogen mycelia infected into united style through stylar fissures, and then spread toward basal style along pluricellular hairs. In 30 days or so after flower fall, an opening sinus formed which connected calyx tube and carpel cavity. In 60 days or so after flower fall, hyphae spread into the carpel cavity along the opening sinus, and colonized fruit tissues through cracks on the carpel wall, causing fruit rot. The results reveal the reasons underlying the high incidence of moldy core disease on Starkrimson Delicious apple, and provide implication for developing new disease control strategy against apple moldy core disease.
Five new species and one species new to China of Lactarius are reported from tropical and subtropical regions of China based on morphological comparisons and analyses of ITS-nrDNA sequences. The new species L. cheilocystidiatus and L. polycystis are distinctive in L. subg. Plinthogalus due to the presence of macrocystidia. They are closely related to Thai L. crassiusculus. Lactarius gloeocarpus and L. vulgaris are new members of L. subg. Lactarius, characterized by yellowish sticky pileus, reticulate spores and large macrocystidia. They form a species complex with Chinese L. pallido-ochraceus, L. pallidizonatus and Indian L. thindii. Lactarius hunanensis is a new species with strong temperate affinity. It is distinguished by the ixolattice pileipellis with olivaceous incrustations and spores with zebroid ornamentation. Lactarius austroscrobiculatus, a tropical species originally described from Indonesia, was reported in China for the first time. The specimens studied were collected from Anhui, Hainan, Henan, Hunan, Jiangxi and Yunnan. Species documented in this study are representative fungi in the Sino-Japan Forest subkingdom.
The distribution and diversity of endophytic fungi in different tissues of Lycium ruthenicum in Xinjiang were analyzed for providing scientific basis of exploring the interaction mechanism between endophytic fungi and their hosts under saline-alkali and drought conditions, and for screening and developing beneficial microbial resources for growth promotion, disease prevention and stress resistance of the host. Community composition and difference of endophytic fungi in different tissues of Lycium ruthenicum were compared by ITS high-throughput sequencing technology, and ecological functions of the fungal community were predicted by FunGuild database. In total, 354 058 high-quality endophytic fungal sequences were obtained, involving 291 OTUs, belonging to 9 phyla, 19 classes, 34 orders, 50 families and 60 genera. The analysis of alpha diversity index showed that diversity and richness of the fungal community in roots and fruits were comparatively high, while that in leaves moderate. The fungal diversity in flowers was the lowest, and the richness in stems was the lowest. Ascomycetes were dominant, and their relative abundances in flowers, leaves, fruits, stems and roots were 86.85%, 72.36%, 75.97%, 84.44% and 85.02%, respectively. Alternaria was predominant, distributing in different tissues including flowers, leaves, fruits, stems and roots, and accounting for 85.41%, 69.79%, 47.07%, 79.94% and 36.97%, respectively. Other comparatively dominant fungi were Cladosporium, Acremonium, Neocamarosporium, Phoma, and Thecaphora. The relative abundance of the tissue-specific genera in each tissue was less than 1%. Many endophytic fungi had the functions of promoting growth and strengthening stress resistance of the host against extreme environments such as saline-alkali and drought. The composition and relative abundance of common and specific fungi in different tissues were quite different. FUNGuild software platform analysis showed that the relative abundance of pathotroph-saprotroph-symbiotroph fungi was the highest, accounting for 85.41%, 69.84%, 47.24%, 79.98% and 37.09% in the flowers, leaves, fruits, stems and roots of L. ruthenicum, respectively. In the fruits and leaves there were some opportunistic pathogens with relative abundance more than 1%. There were more saprophytic fungi in roots. In conclusion, L. ruthenicum contained abundant functional endophytic fungi, which were quite different in composition and ecological function for different tissues. In addition, there were a large number of unidentified species with undefined function, which need to be further investigated.
Epichloë endophytes are widespread symbionts of plants that asymptomatically inhabit aerial host tissues, and not only confer tolerance to biotic and abiotic stresses, but also affect soil microbes. Endophyte-infected (EI) and endophyte-free (EF) Festuca arundinacea are used as experimental materials to investigate the effects of endophyte Epichloë and soil saline-alkali stress on the arbuscular mycorrhizal fungal (AMF) diversity and community composition in roots of tall fescue. A pot experiment under endophyte infection (EI and EF) and saline-alkali stress treatments (0, 200, 400 and 600 mmol/L) was conducted. There are five replicates per treatment. AMF diversity in roots of tall fescue is determined by high throughput sequencing. The diversity of AMF in EF tall fescue roots decreased with the increase of saline-alkali stress treatment level, and the presence of the endophytes alleviated this effect. Under the 200 and 400 mmol/L saline-alkali stress treatments, the endophytes increased the diversity of AMF in tall fescue roots; in addition, the endophytes caused a shift in AMF community composition, reducing the relative abundance of Funneliformis and increasing the relative abundance of Claroideoglomus, Glomus and some unclassified AMF. The results of structural equation model (SEM) showed that the endophytes increased the AMF diversity of tall fescue roots by increasing the concentration of soil total phosphorus under saline-alkali stresses. This study provides reference for selecting the plant-microbial symbionts during ecological restoration in areas with saline-alkali pollution.
Wolfiporia hoelen, one of the traditional Chinese medicines, is simultaneously edible and medicinal. Although it has been cultivated in China for a long time and on a large scale at present, an ambiguous mating system has limited the improvement of cultivated strains. In our previous studies, the homokaryons were discovered, the mating system and life cycle of the fungus were elucidated, the methods of distinguishing the homokaryons based on culture characteristics and molecular markers were established. However, whether the methods can be used in all the W. hoelen population is undefined. In the present study, a new type of homokaryons of W. hoelen derived from Japanese strain NBRC30628 was discovered and identified. The culture characteristics, mating reaction and crossing of these homokaryons were studied. The new type of homokaryons was discriminated through antagonism with parent strain. It was found that there were no differences in growth rates on PDA and sawdust and colony morphology between the homokaryons and parent strain. No mating reaction was discovered in the mating process. The rpb2 marker was used to distinguish the homokaryons and verify the results of mating. Mating test showed that the new type of homokaryons could cross with the homokaryons previously discovered, and the hybrids could produce antagonism reactions with two parent strains. This finding supplements a distinguishing method of identifying homokaryons of W. hoelen population.
The symbiotic characteristics of early stage fungi associated with native and exotic pines were explored. Axenically germinated seedlings of native (Pinus yunnanensis and P. armandii) and exotic (P. greggii and P. maximartinensis) pines were inoculated with spore suspension of Suillus luteus, S. phylopictus and Laccaria vinaceoavellanea in the culture room. Six months later, S. luteus and S. phylopictus ectomycorrhizae were successfully produced on all four tested tree species, and P. armandii showed good receptivity to both Suillus species. However, L. vinaceoavellanea ectomycorrhizae were only found in P. greggii and P. maximartinensis, with a low colonization rate of 14.3%. This is the first report about these ten mycorrhizal combinations, and the mycorrhizae synthesized by the same fungi morphologically and anatomically showed similar characters in all tested tree species. Plant growth affected by these three fungi varied with tree species, and the plant growth of exotic pines was much faster than that of native pines. Although the colonization rate of the Suillus species on P. armandii was the highest, their effects on host growth were not significant. This study suggests that spore inoculation is an economical and effective method to study the mycorrhizal synthesis of early stage fungi such as Suillus and Laccaria species. There were differences between native and exotic pines in mycorrhizal formation with early stage fungi, as well as the effects of formed mycorrhizae on host plant growth.
Funalia trogii is a typical thermotolerant fungus with strong fruiting ability. To clarify the basic characteristics and functions of catalase (CAT) gene family, the identification of CAT in genomes of two monokaryons (Ct001_29 and Ct001_31) with different mating types was conducted, and the CAT expression profiles were examined in mycelia (cultured at different temperatures), primordia, and fruiting bodies. There were three CAT genes (Ctcat1-Ctcat3) in the genome of F. trogii, encoding 510-743 amino acids. The gene structure and sequences of the alleles of Ctcat1, Ctcat2, and Ctcat3 were relatively conserved, but a few SNPs (6-14) existed in their coding regions. CAT activity varied with culture temperature. The maximal CAT activity (278 U/mg protein) was detected in mycelia cultured at 35 °C, and the minimal CAT activity (4 U/mg protein) was detected in primordia. The expression abundance of Ctcat2 was significantly higher than that of Ctcat1 or Ctcat3. Ctcat1 and Ctcat3 had similar expression patterns, showing up-regulated expression in mycelia, primordia, and fruiting bodies. However, Ctcat2 had the opposite expression pattern, showing down-regulated expression in mycelia cultured at 35 °C, primordia, and fruiting bodies. Allelic expression bias was detected in Ctcat2 (in primordia) and Ctcat3 (in mycelia cultured at 25 °C and 35 °C). The expression profiles of Ct29cat2 and Ct29cat3 were both higher than those of their alleles. The allelic expression bias revealed in this study lays the foundation for studying the gene function of macrofungal CAT in the future.
Cordyceps militaris is an important medicinal fungus with various biological functions. In recent years, its ability to accumulate metal ions has received extensive attention by researchers. However, the ability of C. militaris to accumulate Cr3+ and the corresponding physiological responses have not been reported in the literature. In this study, oat media containing different concentrations of Cr3+ were used to culture C. militaris fruit body, and the biomass and content of adenosine, cordycepic acid, ergosterol and Cr3+ in C. militaris fruit body were determined. The results showed that the measured indexes manifested a trend of increasing first and then decreasing with the increase of Cr3+ concentration in the medium, except for ergosterol content which showed a reduction trend. The dry and fresh weight and Cr3+ content of fruit body reached the maximum value when the medium contained 700 mg/L Cr3+, having increased by 36.85%, 35.53% and 202.12% as compared with those of the control, respectively. The cordycepin content and adenosine content reached the maximum value when Cr3+ concentration reached 1 500 mg/L, being 94.61% and 530.29% higher than those of the control, respectively. The cordyceps acid content and polysaccharide content reached the maximum value at Cr3+ concentration of 1 100 mg/L and 1 200 mg/L, respectively, being 123.62% and 21.39% higher than those of the control, respectively. In conclusion, this study revealed for the first time the Cr3+ enrichment ability of C. militaris fruiting body, and the variation of biomass and content of some secondary metabolites in fruiting body under Cr3+ treatment. Further study of Cr3+ enrichment mechanism of C. militaris is needed for laying a foundation of developing chromium ion-rich C. militaris functional products.
The volatile organic compounds (VOCs) of three Tuber species ascocarps were analyzed by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Total 173 VOCs were detected in T. pseudohimalayense, T. sinoaestivum and T. indicum ascocarps, including 11 common compounds and the relative content of 1-octen-3-ol is the highest. Principal component analysis and discriminant analysis on the VOCs also supported the feasibility of identifying the three Tuber species by relative content of the ascocarps VOCs. It will be a useful tool for quality evaluation and identification of processed truffle products.
