First Report of Pseudoidium santalacearum Causing Foliar Powdery Mildew on Santalum album in Guangdong Province, China.

Santalum album Linn is an evergreen and facultative root hemiparasitic tree. It is attached to host roots through haustorium to withdraw water and nutrients. Its aromatic heartwood, honored as "Green Gold", is widely used in perfumes, incense, medicine, and skin cancer prevention (Sandeep and Manohara 2019, Polaiah et al. 2020). From February to June 2022, powdery mildew was observed on over 80% of S. album in the nursery with a mean temperature and relative humidity of 30/22°C (day/night) and 55/82% (day/night) at the Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangdong, China (23°11'N, 113°23'E). Initial symptoms were circular to irregular white colonies on the leaf surface (Fig. 1). As the disease progressed, white mycelia covered the entire leaf surface and premature defoliation of the infected leaf was observed. Then a bare stem remained and subsequently desiccated (Sundararaj et al. 2022). Infected leaves with mycelia were collected and observed under a Zeiss AX10 microscope. Foot cells of conidiophores (n = 30) were straight to slightly curved cylindrical, and the length and width of foot cells were up to 24.8 to 44.6 × 8.2 to 11.7 µm. Conidiophores (n = 30) were straight, 83.5 to 127.3 µm long × 6.6 to 7.6 µm wide and produced three immature conidia in chains. Conidia (n = 30) were elliptical to cylindrical, measured 31.3 to 41.2 µm × 11.3 to 13.7 µm. The sexual stage was not found. The morphological characterization of our samples was consistent with powdery mildew of S. album in India, termed Pseudoidium santalacearum (Patel et al. 2015). However, the molecular identification was not performed. In this study, we carefully collected the mildew and extracted the genomic DNA. The internal transcribed spacer (ITS) region of rDNA was amplified using universal primers ITS1 and ITS4 (White et al. 1990). The ITS sequences of the three samples (GenBank Accession No. OQ746371, OQ746372, and OQ746373) were all more than 99% identical with the sequence of Erysiphe palczewskii on Caragana arborescens (GenBank Accession No. LC010048) (Takamatsu et al. 2015). It is known that Erysiphe containing chasmothecia belongs to teleomorph, whereas Pseudoidium is an asexual morph of Erysiphe, belonging to anamorph (Hsiao et al. 2022, Meeboon and Takamatsu 2016). In our samples, there was no chasmothecia, and the morphological characterization was consistent with P. santalacearum (Patel et al. 2015). Therefore, we classified it as P. santalacearum. Phylogenetic analysis showed that P. santalacearum had a closer relationship with the E. palczewskii (Fig. 2). Pathogenicity tests were conducted by gently pressing the infected leaves onto five healthy S. album plants. Five non-inoculated plants were used as controls. The plants were placed in a growth chamber (Light/Dark: 16 h/8 h, temperature: 25 ± 2°C). Inoculated leaves developed powdery mildew symptoms after 7-12 days, whereas the control plants remained symptomless (Fig. 1). Morphological and molecular characters of the fungus on the inoculated plants were identical to those on naturally infected S. album, fulfilling Koch's postulates. To our knowledge, this is the first report of P. santalacearum on S. album in Guangdong, China. Powdery mildew can generally result in a high mortality of S. album seedlings. Therefore, identification of the pathogen provides a scientific basis for effective diagnosis and prevention of the disease and contribute to an appropriate nursery management of S. album seedlings.

Nitrogen rather than carbon released by litter decomposition mediates nutrient relationships in a multispecies forest plantation with hemiparasite.

Hemiparasitic plants influence community composition by altering nutrient cycling. Although hemiparasites can deplete a host's nutrients via parasitism, their potentially positive effects on nutrient return to multispecies communities remain unclear. We used 13C/15N-enriched leaf litter of the hemiparasite sandalwood (Santalum album, Sa) and two N2-fixing hosts of acacia (Acacia confusa, Ac) and rosewood (Dalbergia odorifera, Do), either as a single-species or mixed-species litter, to elucidate nutrient return by litter decomposition in an acacia-rosewood-sandalwood mixed plantation. We determined litter decomposition rates, litter C and N release, and the resorption of C and N from seven litter types (Ac, Do, Sa, AcDo, AcSa, DoSa, and AcDoSa) at 90, 180, 270, and 360 days. We found that non-additive mixing effects were common during the decomposition of mixed litter and depended on litter type and decomposition timing. After rapidly increasing for around 180 days, both the decomposition rate and release of C and N from litter decomposition declined, but the resorption of litter-released N by the target tree species increased. There was a 90-day lag time between the release and resorption of litter N. Sandalwood litter consistently stimulated the litter mass loss of its mixed litter. Rosewood had the highest release rate of litter 13C or 15N from litter decomposition, but resorbed more litter 15N into its leaves than other tree species. In contrast, acacia had a lower decomposition rate and a higher 15N resorption in its roots. Initial litter quality was closely correlated with the release of litter 15N. Neither the release nor resorption of litter 13C significantly differed among sandalwood, rosewood, and acacia. Our study demonstrates that the fate of litter N, rather than litter C, mediates nutrient relationships in mixed sandalwood plantations and thus provides important silvicultural implications for planting sandalwood with other host species.

Host root exudates initiate a foraging preference by the root parasite Santalum album.

Haustoria of root-parasitic plants draw nutrients from the roots of host species. While recent studies have assessed host preferences of parasitic plants, how root-exuded chemicals can mediate host tropism and selection by root-parasitic plants is poorly understood. Under greenhouse conditions, we performed two pot experiments to determine whether the root parasite Santalum album selectively forages for superior hosts (N2-fixing Acacia confusa Merr. or Dalbergia odorifera T. Chen) rather than for inferior hosts (non-N2-fixing Bischofia polycarpa (levl.) Airy Shaw or Dracontomelon duperreranum Pierre), and whether S. album uses host root exudates and/or specific chemicals in these root exudates to locate and trigger haustorium formation. Lateral roots and haustoria of S. album seedlings exhibited greater growth in the direction of D. odorifera roots than toward roots from the other three hosts. Comparative metabolic analysis revealed that D. odorifera root exudates were enriched in isoflavonoid, flavonoid and flavone/flavonol biosynthesis pathways, and that the relative contents of flavonoids were significantly greater in the root exudates of D. odorifera than in those of the other three hosts. Root exudates from D. odorifera significantly promoted S. album root growth, haustorium formation and reactive oxygen species accumulation in haustoria. Our results demonstrate that the key step in plant parasitism by S. album is based on root exudation by a host plant; the exudates function as a metabolite signal that activate lateral root growth and haustorium formation. Our results also indicate that flavonoids in the root exudates could play an important role in S. album foraging activity. Information on the responses of root parasites to host root exudates and/or haustorium-inducing chemicals may be useful for selecting superior host species to plant with valuable species of root parasites.

First Report of Phytoplasma Belongs to 16SrV Group Associated with Witches'-broom Disease of Taxillus chinensis in China.

Taxillus chinensis (DC.) Danser is a hemiparasitic shrub, widespread in Southern China (Fu et al., 2001). T. chinensis can parasitize a wide range of species (e.g., Camellia spp., Ficus virens and Osmanthus fragrans), which obviously suppressed host growth by robbing nutrient and water through haustorium, causing considerable tree damage. During field visits to Dongguan (22°86'N, 13°97'E) and Guangzhou (23°19'N, 113°31'E), Guangdong Province, in April-July 2021, the typical phytoplasma-suspected symptom manifested as stunting, leaflet, leaf chlorosis and witches'-broom were observed in almost 36% of T. chinensis plants. Leaf samples were collected from six randomly collected plants with symptoms and six symptomless plants (Fig 1). Among them, half of T. chinensis plants parasitized on the host Elaeocarpus sylvestris, the other half on the O. fragrans. No apparent symptoms were observed on both two host plants. Total DNA was extracted from 0.5 g fresh leaf of T. chinensis plants with and without symptoms, as well as two host plants E. sylvestris and O. fragrans, using the CTAB method (Doyle et al., 1990). Nested polymerase chain reactions (PCRs) were performed on DNA extracts of all tested plants with primer pairs of P1/P7 and R16mF2/R16mR1 for 16S rRNA gene (Lee et al., 1993) and rp(v)F1/rpR1 for rp gene (Lee et al., 1998). All amplicons were obtained from symptomatic samples of T. chinensis and host plant E. sylvestris, whereas no such products resulted from DNAs of symptomless plants and O. fragrans. The amplicons were purified and sequenced by Sanger method (Rui Biotech, Guangzhou, China). The amplicon of 16S rRNA and rp genes is 1346 bp and 938 bp, respectively. BLAST comparison of the 16S rRNA (accession no. OL412744) and rp (accession no. OL473789) sequences of the T. chinensis witches'-broom phytoplasma yielded 99.6% sequence identity with those of phytoplasmas of group 16SrV jujube witches'-broom (JWB) phytoplasma (accession no. CP025121 for 16S rRNA gene and AF396941 for rp gene). The 16S rRNA gene sequence of phytoplasma in host plant E. sylvestris (accession no. OM885990) is 99.7% similarity to the 'Elaeocarpus zollingeri' yellows phytoplasma (accession no. LC257960) and 99.4% similarity to the 'Elaeocarpus sylvestris' decline phytoplasma (accession no. MW553140), but 95.8% similarity to the 16S rRNA gene of phytoplasma in T. chinensis. The virtual RFLP tool, iPhyClassifier delineated the T. chinensis phytoplasma (accession no. OL412744) to group 16SrV-B (accession no. AB052876) with the similarity coefficient 1.0 (Fig 2), and phytoplasma in E. sylvestris to group 16Sr group XXXII with the similarity coefficient 0.97. Phylogeny analyses of 16S rRNA and rp genes (MEGA version 7.0.14, USA) using reference phytoplasmas from GenBank confirmed sequencing results and placed the T. chinensis phytoplasma in group 16SrV-B (Fig. 3 and 4). In China, the 16SrV-B phytoplasma group has been reported in Amaranthus retroflexus (Yang et al., 2011), Liriodendron chinense (Li et al., 2012), Prunus salicina (Gao et al., 2020) and sweet potato (Li et al., 2021). This is the first report of a 'Ca. Phytoplasma ziziphi', 16SrV-B related phytoplasma associated with parasitic T. chinensis in China. The results of this study indicate that T. chinensis could be a vector to spread phytoplasmas 16SrV group through parasitism and this can be helpful for related research.

The yellowhorn AGL transcription factor gene XsAGL22 contributes to ABA biosynthesis and drought tolerance in poplar.

Regulation of abscisic acid (ABA) biosynthesis helps plants adapt to drought stress, but the underlying molecular mechanisms are largely unclear. Here, a drought-induced transcription factor XsAGL22 was isolated from yellowhorn (Xanthoceras sorbifolium Bunge). Yeast one-hybrid and electrophoretic mobility shift assays indicated that XsAGL22 can physically bind to the promoters of the ABA biosynthesis-related genes XsNCED6 and XsBG1, and a dual-luciferase assay showed that XsAGL22 activates the promoters of the later two genes. Transient overexpression of XsAGL22 in yellowhorn leaves also increased the expression of XsNCED6 and XsBG1 and increased cellular ABA levels. Finally, heterologous overexpression of XsAGL22 in poplar increased ABA content, reduced stomatal aperture and increased drought resistance. Our results suggest that XsAGL22 is a powerful regulator of ABA biosynthesis and plays a critical role in drought resistance in plants.

Bacterial Community Selection of Russula griseocarnosa Mycosphere Soil.

Russula griseocarnosa is a wild, ectomycorrhizal, edible, and medicinal fungus with high economic value in southern China. R. griseocarnosa fruiting bodies cannot be artificially cultivated. To better understand the effects of abiotic and biotic factors on R. griseocarnosa growth, the physicochemical properties of R. griseocarnosa and its associated bacterial communities were investigated in two soil types (mycosphere and bulk soil) from Fujian, Guangdong, and Guangxi Provinces. The results revealed that the diversity, community structure, and functional characteristics of the dominant mycosphere bacteria in all geographical locations were similar. Soil pH and available nitrogen (AN) are the major factors influencing the mycosphere-soil bacterial communities' structure. The diversity of soil bacteria is decreased in R. griseocarnosa mycosphere when compared with the bulk soil. Burkholderia-Paraburkholderia, Mycobacterium, Roseiarcus, Sorangium, Acidobacterium, and Singulisphaera may also be mycorrhiza helper bacteria (MHB) of R. griseocarnosa. The functional traits related to the two-component system, bacterial secretion system, tyrosine metabolism, biosynthesis of unsaturated fatty acids, and metabolism of cofactors and vitamins were more abundant in R. griseocarnosa mycosphere soil. The mycosphere soil bacteria of R. griseocarnosa play a key role in R. griseocarnosa growth. Application of management strategies, such as N fertilizer and microbial fertilizer containing MHB, may promote the conservation, propagation promotion, and sustainable utilization of R. griseocarnosa.

Whole genome sequencing and genome annotation of the wild edible mushroom, Russula griseocarnosa.

Russula griseocarnosa is a species of edible ectomycorrhizal fungi with medicinal properties that grows in southern China. Total DNA was isolated from a fresh fruiting body of R. griseocarnosa and subjected to sequencing using Illumina Hiseq with the PacBio RS sequencing platform. Here, we present the 64.81 Mb draft genome map of R. griseocarnosa based on 471 scaffolds and 16,128 coding protein genes. The gene annotation of protein coding genes was used to obtain corresponding annotations by blastp. Phylogenetic analysis revealed a close evolutionary relationship of R. griseocarnosa to Heterobasidion irregulare and Stereum hirsutum in the core Russulales clade. The R. griseocarnosa genome encodes a repertoire of enzymes engaged in carbohydrate and polysaccharide metabolism, along with cytochrome P450s and secondary metabolite biosynthesis. The genome content of R. griseocarnosa provides insights into the genetic basis of its reported medicinal properties and serves as a reference for comparative genomics of fungi.

Fungal Systematics and Evolution: FUSE 5.

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

Co-existence of Rhizobia and Diverse Non-rhizobial Bacteria in the Rhizosphere and Nodules of Dalbergia odorifera Seedlings Inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium-Like and Burkholderia pyrrocinia-Like Strains.

Rhizobia induce root nodules and fix atmospheric N2 for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume-bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of Dalbergia odorifera seedlings that were uninoculated or inoculated with Bradyrhizobium elkanii H255, Rhizobium multihospitium-like HT221, or Burkholderia pyrrocinia-like H022238, in two growth media (nitrogen [N]-supplied soil or N-omitted potting mix). Seedlings inoculated with Bradyrhizobium had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the 15N natural abundance method, it was shown that the inoculated plants had significantly higher N2 fixation efficiency (48-57%) and specific nodule activity [269-313 µg N mg-1 of dry weight (dwt) nodule] compared to the uninoculated plants (203 µg N mg-1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (P < 0.05), with the exception of the inoculated soil rhizospheres. Regarding non-rhizobial bacteria, three genera, i.e., Lactococcus, Bacillus, and Pseudomonas, were consistently enriched in the rhizosphere and Bradyrhizobium, Chloroplast norank (which belongs to Cyanobacteria), and Lactococcus were commonly found in the nodules. In contrast, common rhizobial genera (including Rhizobium, Mesorhizobium, and Burkholderia) were only present in the nodules at low relative abundances (0.01-3.41%). Regarding non-rhizobial bacteria, 32 genera were found in the nodules, with non-rhizobial bacteria being predominant in the N omitted potting mix (with a relative abundance of 56-87%). This study suggests that legume nodules are inhabited by a high diversity of non-rhizobial bacteria, which may play a vital role in nodulation and N2 fixation in the host plants.

[Biodiversity of Rhizobia associated with Acacia melanoxylon grown in South China].

OBJECTIVE: This study aimed to investigate the genetic diversity of 174 isolates of symbiotic bacteria associated with Acacia melanoxylon obtained from 15 sampling sites in Guangdong, Fujian and Jiangxi provinces of China. METHODS: The 16S rDNA restriction fragment length polymorphism (RFLP) and phylogenetic analyses of the 16S rDNA and housekeeping genes (recA, glnII and atpD). RESULTS: In the 16S rDNA PCR-RFLP analysis, 9 rDNA types were identified among the 174 isolates; Phylogenetic analyses based on 16S rDNA and housekeeping gene sequences indicated that 34 representative isolates belonged to the genus Bradyrhizobium, Rhizobium, Mesorhizobium in Alpha-Proteobacteria, and the most closely related strains are Bradyrhizobium liaoningense, Bradyrhizobium betae, Bradyrhizobium cytisi, Rhizobium multihospitium and Mesorhizobium plurifarium. CONCLUSION: All of the isolates could be identified to general, and Bradyrhizobium, Rhizobium or Mesorhizobium could be the dominant microsymbiont. The microsymbionts associated with Acacia melanoxylon showed relative genetic diversity.

[Genotypic analysis and plant growth-promoting ability of four plant growth-promoting bacteria from mangrove].

OBJECTIVE: We identified four strains of plant growth-promoting bacteria (PGPB) and their plant growth-promoting ability. METHODS: Four PGPB strains were genetically analyzed by PCR detection of nifH and 16S rRNA gene. Phosphate-solubilizing and nitrogen-fixation capacity were examined by spectrophotometric quantification and acetylene reduction assay, respectively. Effect of strain inoculation on plant growth was also evaluated. RESULTS: Phylogenetic analysis based on nifH and 16S rRNA gene sequences indicated that strain HN011 was mostly related to Vibrio natriegens, and SZ7-1 and SZ7-2 resembled Klebsiella oxytoca. Although similarity of 16S rRNA sequence showed that SZ002 belongs to Paenibacillus sp., nifH gene of SZ002 had high sequence similarity with Klebsiella genus. Phosphate solubilization showed that insoluble phosphate was well solubilized in the liquid medium by all four strains of PGPB, which also had high nitrogen-fixation capacity. Plant dry weight, total N and total P were higher in some inoculated than in the non-inoculated plants (P < 0.05). CONCLUSION: Our results showed that all four strains of PGPB isolated from mangrove had both phosphate solubilization and nitrogen fixation ability, resulting in beneficial effects on growth.