First Report of Anthracnose Crown Rot on Strawberry (Fragaria × ananassa Duch.) Caused by Colletotrichum pandanicola in Yunnan Provence, China.

Strawberry (Fragaria × ananassa Duch.), a widely grown octoploid species, is one of the most important economic fruit crops and has been widely cultivated in the world, including China. In December 2021, a serious crown rot disease (approximately 50% incidence) was observed in strawberry (cultivar Miaoxiang) plantations in Qujing City, Yunnan Province, China. Symptoms observed on aboveground part withered rapidly, reddish-brown marbled necrosis on crown. The roots were healthy and strong, but the plants finally died. To isolate the causal agent of this disease, crown tissues from five strawberry plants showing typical symptoms were cut into pieces of 5×5 mm, and the pieces were surface-sterilized with 75% ethanol for 45 s followed by 2.5% NaClO for 3 min and rinsed thrice with sterile water, and then placed onto potato dextrose agar (PDA) for 7 days at 25 ºC. After 3 to 4 days, extended single hyphal tips from the tissues were transferred to PDA and incubated for 7 days at 25 ºC. The colonies were initially white, later became somewhat zonate, velvety, cyan gray on the upper side and cyan ink pigment ring on the reverse side of plates, with concentric rings of salmon sporodochia. Many yellowish or orange creamy conidial droplets formed on PDA after 14 days at 25 ºC. Fifty-nine isolates were obtained, and three isolates QLYRR1, QLMCR9, and QLMCR39 were selected for further experiments. Conidia were hyaline, cylindrical with rounded ends, 12.17-19.35×3.71-6.30 µm (average±SD, 15.24±1.37×5.09±0.45 µm, n=150), L/W ratio = 2.99. The three isolates were molecularly identified using the genomic regions of internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes, and the sequences were deposited in GenBank (accession nos. QLYRR1, QLMCR9, QLMCR39: ON668272, ON668256, ON668257[ITS], ON684302, ON684300, ON684301[ACT], ON684316, ON684314, ON684315[CHS-1], ON684292, ON684290, ON684291[GAPDH], ON684286, ON684284, ON684285[TUB2]). The phylogenetic analysis of experimental strains was performed by Maximum-likelihood (ML) tree and Bayesian inference (BI) method. Nucleotide sequences exhibited three isolates were clustered with the ex-type strain C. pandanicola strain MFLUCC 170571T found in Thailand, C. pandanicola strains (SAUCC201152, SAUCC200204) found in Shandong Province, and the holotype stain C. parvisporum YMF 1.06942T found in Guangxi Province, China. Morphologically, isolates were easily distinguished from C. parvisporum by the colony on PDA and the size of conidia (Yu et al. 2022). Morphological characteristics and phylogenetic analyses revealed that QLYRR1, QLMCR9, and QLMCR39 belong to C. pandanicola, the members of the C. gloeosporioides species complex (Tibpromma et al. 2018; Mu et al. 2021). Koch's postulates were tested by strawberry plants (two cultivars, Akihime and Miaoxiang) in vivo, strawberry plants were tested for the three isolates by spraying 1×106 conidia/mL suspension on three seedlings. Three seedlings sprayed with sterile distilled water were served as control. All of the plants were transferred to a glasshouse with a 28/20 °C day/night temperature range and natural sunlight. After 6 weeks, QLYRR1-, QLMCR9-, and QLMCR39-sprayed seedlings were stunted and developed typical wilt symptoms similar to those observed in the field with the incidence for 3, 3, and 3 seedlings, respectively. The negative control remained asymptomatic. The fungi were reisolated again from lesions of diseased plants and leaves with 100% frequency, and morphological characteristics and tested gene sequences were identical to the original isolates in this note, thus fulfilling Koch's postulates. C. pandanicola was described from the healthy leaves of Pandanus sp. and the lesion fruits of Juglans regia. To our knowledge, this is the first report confirming C. pandanicola causes anthracnose crown rot on strawberries in China. C. pandanicola has the potential for causing serious losses to the strawberry industry, and research is needed on management strategies to minimize losses.

Comparison of the Bulk and Rhizosphere Soil Prokaryotic Communities Between Wild and Reintroduced Manglietiastrum sinicum Plants, a Threatened Species with Extremely Small Populations.

Huagaimu (Manglietiastrum sinicum) trees are critically endangered species and classified as a plant species with extremely small populations in China. Rhizospheres and bulk soils prokaryotic communities play an important role to protect and promote plants health and growth. However, the compositions and structures of prokaryotic communities in wild and reintroduced M. sinicum rhizospheres and bulk soils are still poorly understood. In the present study, prokaryotic communities in wild and reintroduced M. sinicum rhizospheres and bulk soils were compared using high-throughput sequencing. Thirty-two phyla, 76 classes, 193 orders, 296 families, and 470 genera of prokaryotes were obtained. Proteobacteria and Acidobacteria were the two most abundant phyla in all soil samples. The compositions and structures of prokaryotic communities were overall similar, and the abundance of some taxa varied significantly among soil samples. Soil prokaryotic communities were significantly affected by soil pH, total nitrogen, total phosphorus, and total potassium. Eleven of predicted functions were significantly different among the four soil groups. This study provides for the first insights into the compositions, structures, and potential functions of prokaryotic communities associated with wild and reintroduced M. sinicum rhizospheres and bulk soils, and providing a foundation for future research to help protect this endangered species.

Rhizospheric soil fungal community patterns of Duchesnea indica in response to altitude gradient in Yunnan, southwest China.

The magnitude of the impact of altitude gradient on microbial community and diversity has been studied in recent decades. Whereas bacteria have been the focus of most studies, fungi have been given relatively less attention. As a vital part of the macro- and microscopic ecosystem, rhizosphere fungi play a key role in organic matter decomposition and relative abundance of plant species and have an impact on plant growth and development. Using Duchesnea indica as the host plant, we examined the rhizosphere soil fungal community patterns across the altitude gradient in 15 sites of Yunnan province by sequencing the fungal ITS2 region with the Illumina MiSeq platform. We determined the fungal community composition and structure. We found that, surprisingly, rhizosphere soil fungal diversity of D. indica increased with altitudinal gradient. There was a slight difference in diversity between samples from high- and medium-altitude sites, with medium-altitude sites having the greater diversity. Furthermore, the rhizosphere soil fungal community composition and structure kept changing along the altitudinal gradient. Taxonomic results showed that the extent of phylum diversity was greatest at high-altitude sites, with Ascomycota, Basidiomycota, Zygomycota, and Glomeromycota as the most dominant fungal phyla.

Comparison of the Rhizosphere Soil Microbial Community Structure and Diversity Between Powdery Mildew-Infected and Noninfected Strawberry Plants in a Greenhouse by High-Throughput Sequencing Technology.

The aim of this study was to compare the microbial community structure and diversity in powdery mildew-infected and noninfected strawberry plant rhizosphere soils in the greenhouse based on variations in the 16S rRNA gene V3-V4 and fungal ITS2 regions by Illumina amplicon sequencing. Powdery mildew infection reduced the number of operational taxonomic units (OTUs) and prokaryotic and fungal community richness/diversity indexes in the rhizosphere soils compared with those in healthy plant soils. Furthermore, 3543 prokaryotic and 581 fungal OTUs were obtained at the 97% similarity level. Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi were the dominant bacterial phyla; Woesearchaeota_DHVEG-6, Bathyarchaeota, and Thaumarchaeota were the dominant archaea; and Ascomycota, Basidiomycota, unclassified_fungi, and Zygomycota were the dominant fungal phyla. Their proportions differed significantly among samples. Wolbachia, Devosia, Pseudolabrys, Streptomyces, and Rhizomicrobium were the most abundant bacterial genera; their proportions differed significantly among samples. Most Pseudomonas, Streptomyces, and 'norank' group members might be potential antagonistic microorganisms of powdery mildew pathogens, and Wolbachia and Rickettsia might be pathogen-transmitting vectors. Microascus, Clitopilus, and Ciliophora were the dominant fungi, and their community structures and abundances significantly differed among samples. Microascus, Talaromyces, Zopfiella, and Cryptococcus were relatively more abundant in the powdery mildew-infected strawberry plant rhizosphere soils. Fusarium, Trichoderma, Clitopilus, and 'unclassified' group members may be potential antagonistic populations. The results suggested that powdery mildew-infected strawberry fruits and plants cannot be consumed. This report is the first study to illustrate differences in the rhizosphere soil prokaryotic and fungal communities between powdery mildew-infected and noninfected strawberry plants in a greenhouse.

Study of Endogenous Viruses in the Strawberry Plants.

Endogenous viral elements (EVEs) have been reported to exist widely in the genomes of eukaryotic organisms, and they are closely associated with the growth, development, genetics, adaptation, and evolution of their hosts. In this study, two methods-homologous sequence search and genome alignment-were used to explore the endogenous viral sequences in the genomes of Fragaria species. Results revealed abundant endogenous pararetroviruses (EPRVs) in the genomes of Fragaria species, including 786 sequences belonging to five known taxa such as Caulimovirus and other unclassified taxa. Differences were observed in the detected EPRVs between the two methods, with the homologous sequence search having a greater number of EPRVs. On the contrary, genome alignment identified various types and sources of virus-like sequences. Furthermore, through genome alignment, a 267-bp sequence with 95% similarity to the gene encoding the aphid-transmitted protein of Strawberry vein banding virus (Caulimovirus venafragariae) was discovered in the F. chiloensis genome, which was likely a recent insertion. In addition, the statistical analysis of the genome alignment results indicated a remarkably higher abundance of virus-like sequences in the genomes of polyploid strawberries compared with diploid ones. Moreover, the differences in virus-like sequences were observed between the genomes of Fragaria species and those of their close relatives. This study enriched the diversity of viruses that infect strawberries, and laid a theoretical foundation for further research on the origin of endogenous viruses in the strawberry genome, host-virus interactions, adaptation, evolution, and their functions.

Comparative analysis of the microbiomes of strawberry wild species Fragaria nilgerrensis and cultivated variety Akihime using amplicon-based next-generation sequencing.

Fragaria nilgerrensis is a wild strawberry species widely distributed in southwest China and has strong ecological adaptability. Akihime (F. × ananassa Duch. cv. Akihime) is one of the main cultivated strawberry varieties in China and is prone to infection with a variety of diseases. In this study, high-throughput sequencing was used to analyze and compare the soil and root microbiomes of F. nilgerrensis and Akihime. Results indicate that the wild species F. nilgerrensis showed higher microbial diversity in nonrhizosphere soil and rhizosphere soil and possessed a more complex microbial network structure compared with the cultivated variety Akihime. Genera such as Bradyrhizobium and Anaeromyxobacter, which are associated with nitrogen fixation and ammonification, and Conexibacter, which is associated with ecological toxicity resistance, exhibited higher relative abundances in the rhizosphere and nonrhizosphere soil samples of F. nilgerrensis compared with those of Akihime. Meanwhile, the ammonia-oxidizing archaea Candidatus Nitrososphaera and Candidatus Nitrocosmicus showed the opposite tendencies. We also found that the relative abundances of potential pathogenic genera and biocontrol bacteria in the Akihime samples were higher than those in the F. nilgerrensis samples. The relative abundances of Blastococcus, Nocardioides, Solirubrobacter, and Gemmatimonas, which are related to pesticide degradation, and genus Variovorax, which is associated with root growth regulation, were also significantly higher in the Akihime samples than in the F. nilgerrensis samples. Moreover, the root endophytic microbiomes of both strawberry species, especially the wild F. nilgerrensis, were mainly composed of potential biocontrol and beneficial bacteria, making them important sources for the isolation of these bacteria. This study is the first to compare the differences in nonrhizosphere and rhizosphere soils and root endogenous microorganisms between wild and cultivated strawberries. The findings have great value for the research of microbiomes, disease control, and germplasm innovation of strawberry.

Comparative Analysis of the Microbial Community Structures Between Healthy and Anthracnose-Infected Strawberry Rhizosphere Soils Using Illumina Sequencing Technology in Yunnan Province, Southwest of China.

Anthracnose caused by Colletotrichum spp. was widespread in recent years and resulted in great damage to strawberry production. Soil microbial communities were key contributors to host nutrition, development, and immunity; however, the difference between the microbial communities of healthy and anthracnose-infected strawberry rhizosphere soils remains unclear. In this study, the Illumina sequencing technique was used to comparatively study the prokaryotic and fungal community compositions and structures between healthy and anthracnose-infected strawberry rhizosphere soils in Yuxi, Yunnan Province. Both microbial community diversities and richness of anthracnose-infected strawberry rhizosphere soils were higher than those of healthy strawberry rhizosphere soils. A total of 2,518 prokaryotic and 556 fungal operational taxonomic units (OTUs) were obtained at the 97% similarity threshold. Proteobacteria, Thaumarchaeota, and Acidobacteria were the dominant prokaryotic phyla; Ascomycota, unclassified_k__Fungi, and Mortierellomycota were the dominant fungal phyla. The relative abundances of beneficial bacterial phyla Actinobacteria and Firmicutes, genera Streptomyces, Azospirillum, and Bacillus were significantly reduced in anthracnose-infected strawberry rhizosphere soils; the relative abundance of beneficial fungal species Trichoderma asperellum shows a similar tendency with bacterial abundance. Besides Colletotrichum, 15 other potential fungal pathogen genera and seven fungal pathogen species were identified; among the potential pathogen genera and species, eight pathogen genera and Fusarium oxysporum showed significant differences between healthy and anthracnose-infected strawberry rhizosphere soils. The results suggested that strawberry planted in this area may be infected by other fungal pathogens except for Colletotrichum spp. Our present research will provide theoretical basis and data reference for the isolation and identification of strawberry pathogens and potential probiotics in future works.

Comparative Analysis of Fungal Diversity in Rhizospheric Soil from Wild and Reintroduced Magnolia sinica Estimated via High-Throughput Sequencing.

Magnolia sinica is a critically endangered species and considered a "plant species with extremely small populations" (PSESP). It is an endemic species in southeastern Yunnan Province, China, with reproductive barriers. Rhizosphere fungi play a crucial role in plant growth and health. However, the composition, diversity, and function of fungal communities in wild and reintroduced M. sinica rhizospheres remain unknown. In this study, Illumina sequencing of the internal transcribed spacer 2 (ITS2) region was used to analyze rhizospheric soil samples from wild and reintroduced M. sinica. Thirteen phyla, 45 classes, 105 orders, 232 families, and 433 genera of fungi were detected. Basidiomycota and Ascomycota were dominant across all samples. The fungal community composition was similar between the wild and reintroduced rhizospheres, but the fungal taxa relative abundances differed. The fungal community richness was higher in the reintroduced rhizosphere than in the wild rhizosphere, but the diversity showed the opposite pattern. Soil nutrients and leaf litter significantly affected the fungal community composition and functional diversity. Here, the composition, structure, diversity, and ecological functions of the fungal communities in the rhizospheres of wild and reintroduced M. sinica were elucidated for the first time, laying a foundation for future research and endangered species protection.