Infection of the Asian gray shrew Crocidura attenuata (Insectivora: Soricidae) with Sarcocystis attenuati n. sp. (Apicomplexa: Sarcocystidae) in China

Background Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host. Methods Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed. Results Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3–4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9–16.7 × 9.2–10.6 μm with a prepatent period of 10–11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaiatana (i.e., 97.6–98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati. Conclusions Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystisattenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystisscandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future. Graphical abstract

Efficacy of the entomopathogenic nematode isolate Heterorhabditis taysearae to control the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

Background Entomopathogenic nematodes (EPNs) are promising alternatives since they have many characteristics as a biological control agent against insect pests. Results Among indigenous EPN isolated, adapted to local environmental conditions by employing the Galleria baiting strategy, only one sample was positive for the presence of EPNs. The new isolate was identified at the species level using DNA sequencing of the internal transcribed spacer region and the Basic Local Alignment Search Tool search of GenBank showed that the isolate had a high similarity (99%) with that sequence available for Heterorhabditis taysearae. The pathogenicity of the EPN isolate was tested against the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), using different concentrations (60, 90, 120 and 150 IJs/larvae). Data showed that 150 IJs/larvae caused 100% mortality rate, followed by 120 IJs/larvae (90%), while 60 IJs/larvae showed the lowest rate (60%) after 72 h. Conclusions The present study indicated that the native isolate of EPN could be recommended against S. littoralis as an efficient tool in its control programs.

Identification of Monilia species in Tibet and characterization of M. yunnanensis in China

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region (ITS) sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although above-mentioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographical environments did not play a major role in the evolution of M. yunnanensis.

Cold Acclimation in Brachypodium Is Accompanied by Changes in Above-Ground Bacterial and Fungal Communities

Shifts in microbiota undoubtedly support host plants faced with abiotic stress, including low temperatures. Cold-resistant perennials prepare for freeze stress during a period of cold acclimation that can be mimicked by transfer from growing conditions to a reduced photoperiod and a temperature of 4 °C for 2–6 days. After cold acclimation, the model cereal, Brachypodium distachyon, was characterized using metagenomics supplemented with amplicon sequencing (16S ribosomal RNA gene fragments and an internal transcribed spacer region). The bacterial and fungal rhizosphere remained largely unchanged from that of non-acclimated plants. However, leaf samples representing bacterial and fungal communities of the endo- and phyllospheres significantly changed. For example, a plant-beneficial bacterium, Streptomyces sp. M2, increased more than 200-fold in relative abundance in cold-acclimated leaves, and this increase correlated with a striking decrease in the abundance of Pseudomonas syringae (from 8% to zero). This change is of consequence to the host, since P. syringae is a ubiquitous ice-nucleating phytopathogen responsible for devastating frost events in crops. We posit that a responsive above-ground bacterial and fungal community interacts with Brachypodium’s low temperature and anti-pathogen signalling networks to help ensure survival in subsequent freeze events, underscoring the importance of inter-kingdom partnerships in the response to cold stress.

Asproinocybaceae fam. nov. (Agaricales, Agaricomycetes) for Accommodating the Genera Asproinocybe and Tricholosporum, and Description of Asproinocybe sinensis and Tricholosporum guangxiense sp. nov

Asproinocybe and Tricholosporum are not well known, and their placement at the family level remains undetermined. In this study, we conducted molecular phylogenetic analyses based on nuc rDNA internal transcribed spacer region (ITS) and nuc 28S rDNA (nrLSU), and a dataset with six molecular markers (ITS, LSU, RNA polymerase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), 18S nuclear small subunit ribosomal DNA (nrSSU), and translation elongation factor 1-alpha (TEF1-α)) using Bayesian (BA) and Maximum Likelihood (ML) methods, we found that the species of Asproinocybe and Tricholosporum formed an independent family-level clade (0.98/72). Asproinocybaceae fam. nov., a new family, is established here for accommodating this clade. Two new species, Asproinocybe sinensis and Tricholosporum guangxiense, from subtropical and tropical karst areas of China, are also described here.

Morphoanatomical and phylogenetic characterization of the ectomycorrhiza between Laccaria squarrosa with Pinus pseudostrobus and its relevance for reforestation programs

Background: Pinus (Coniferophyta) and Laccaria (Basidiomycota) establish ectomycorrhizal symbioses in natural forests. However, their detailed morphoanatomical and phylogenetic characterization have received little attention. Accurate identification of native host symbionts is of paramount relevance to the production of mycorrhized seedlings for successful reforestation programs. Questions/Objective: We aimed to determine if L. squarrosa is able to establish ectomycorrhizal symbiosis with gymnosperms, thereby widening its host range and highlighting its relevance as a potential inoculant for pine seedlings. Currently, L. squarrosa is only known from its type collection associated with the angiosperm Fagus grandifolia var. mexicana. Studied species: The fungus L. squarrosa and Pinus pseudostrobus, a tree endemic to Mexico. Study site and dates: A Pinus-Quercus forest in Piedra Canteada, Nanacamilpa, Tlaxcala; 2018-2020. Methods: L. squarrosa basidiomata were identified and ectomycorrhizal roots were collected and morphoanatomically characterized. For molecular identification, DNA was extracted, PCR was performed targeting the nuclear ribosomal internal transcribed spacer region (nucrDNA ITS) for the mycobiont identification and the chloroplastic single-locus trnL region for the phytobiont. Results: In the phylogenetic analyses, our sequences from basidiomata and ectomycorrhizae clustered together with L.squarrosa with high values of supporting identity. Meanwhile, P. pseudostrobus was molecularly identified as the phytobiont. Conclusions: This is one of the few worldwide characterizations of Laccaria ectomycorrhiza under field conditions and contributes to the understanding of the ecology, distribution, and economic relevance of the symbiotic association. Our data suggest that L. squarrosa has potential for use as a native inoculant for P. pseudostrobus tree production. Translate stop Translate stop

Multilocus Genotyping and Intergenic Spacer Single Nucleotide Polymorphisms of Amylostereum areolatum (Russulales: Amylostereacea) Symbionts of Native and Non-Native Sirex Species

Sirex noctilio along with its mutualistic fungal symbiont, Amylostereum areolatum (a white rot fungus), is an invasive pest that causes excessive damage to Pinus plantations in Northeast China. In 2015, S. noctilio were found to attack Pinus sylvestris var. mongolica, and often share larval habitat with the native woodwasp, S. nitobei. The objective of this study was to determine the possible origin(s) of the introduced pest complex in China and analyse the genetic diversity between A. areolatum isolated from invasive S. noctilio, native S. nitobei and other woodwasps collected from Europe (native range) and other countries. Phylogenetic analyses were performed using the intergenic spacer (IGS) dataset and the combined 4-locus dataset (the internal transcribed spacer region (ITS), translation elongation factor alpha 1 (tef1), DNA-directed ribosomal polymerase II (RPB2), and mitochondrial small subunit (mtSSU)) of three Amylostereum taxa. The multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed at least three distinct multilocus genotypes (MLGs) of the fungus associated with invasive S. noctilio populations in Northeast China, which may have come from North America or Europe. The IGS region of A. areolatum carried by S. noctilio from China was designated type B1D2. Our results showed a lack of fidelity (the paradigm of obligate fidelity to a single fungus per wasp species) between woodwasp hosts and A. areolatum. We found that the native S. nitobei predominantly carried A. areolatum IGS-D2, but a low percentage of females instead carried A. areolatum IGS-B1D2 (MLG A13), which was presumably due to horizontal transmission from S. noctilio, during the sequential use of the same wood for larval development. The precise identification of the A. areolatum genotypes provides valuable insight into co-evolution between Siricidae and their symbionts, as well as understanding of the geographical origin and history of both Sirex species and their associated fungi.

Designation of the neotype of Triatoma dimidiata (Latreille, 1811) (Hemiptera, Reduviidae, Triatominae), with full integrated redescription including mitogenome and nuclear ITS-2 sequences

The taxonomic status of Triatoma dimidiata (Latreille, 1811) is, by far, the most discussed within Triatominae. Molecular studies have recovered at least three independently evolving lineages in T. dimidiata across its range. The original description of T. dimidiata (as Reduvius dimidiatus) included few taxonomic characters, and no types were assigned. To define and describe the cryptic diversity within T. dimidiata sensu lato (s.l.), a neotype must be designated. For this purpose, all 199 specimens identified as T. dimidiata from the collections of the Smithsonian Institution – National Museum of Natural History and the American Museum of Natural History, ranging from Peru to Mexico, were studied. Only one specimen (from Tumbes, Peru) matched the combination of characters as listed in the original description, and it is herein formally designated as the neotype for T. dimidiata. The neotype is morphologically described and DNA sequences of its whole mitochondrial genome and the nuclear second internal transcribed spacer region (ITS2), commonly used in triatomine molecular systematics studies, are presented and compared to other publicly available sequences of T. dimidiata s.l. in GenBank. Our results suggest that T. dimidiata sensu stricto (s.s.) is somewhat rare and, therefore, unlikely to serve as a major vector of Chagas disease.

First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05′, E119°38′), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 μm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 μm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.

Taxonomy and Phylogeny Reveal Two New Potential Edible Ectomycorrhizal Mushrooms of Thelephora from East Asia

The ectomycorrhizal basidiomycetes genus Thelephora has been understudied in subtropical ecosystems. Many species of Thelephora are important edible and medicinal fungi, with substantial economic value. Two new Thelephora species, T. grandinioides and T. wuliangshanensis spp. nov. are proposed here based on a combination of morphological features and molecular evidence. Thelephora grandinioides is characterized by laterally stipitate basidiocarps with a grandinoid hymenial surface, a monomitic hyphal system with clamped generative hyphae, and the presence of tubular and septated cystidia and subglobose to globose basidiospores measuring as 5.3–7.4 × 4–6.5 µm. Thelephora wuliangshanensis is characterized by infundibuliform basidiocarps, radially black striate on the pileus, a smooth, umber to coffee hymenial surface, a monomitic hyphal system with thick-walled generative hyphae, and basidiospores that turn greenish grey to buff in 5% KOH. Phylogenetic analyses of rDNA internal transcribed spacer region (ITS) and nuclear large subunit region (nrLSU) showed that the two new Thelephora are phylogenetically distinct: T. grandinioides is sister to T. aurantiotincta and T. sikkimensis, while T. wuliangshanensis is sister to a clade comprising T. austrosinensis and T. aurantiotincta with high support as well.