Calmodulin Gene Research Articles

Transcriptome analysis provides insights into the function of the cholinergic system in pearl oyster transplantation immunity

Excessive immune response of pearl oyster after transplantation is the main factor that causes its nucleus rejection and death. In this study, to clarify how the cholinergic system regulates the immune response after transplantation, ACh and mecamylamine hydrochloride (nAChR inhibitor) were used to activate or block the cholinergic system of pearl oysters before transplantation. Transcriptomes of hemocytes were constructed at different times (6, 12, 24, and 48 h) after transplantation. Transcriptome analysis showed that the Kyoto Encyclopedia of Genes and Genomes pathway related to “inositol phosphate metabolism” pathway and Gene Ontology (GO) terms “calcium-ion binding” were significantly enriched in the differential expression genes of the two comparisons, and calmodulin genes were upregulated at 12–24 h after activation of the cholinergic system. Further experiments showed that ACh treatment significantly induced intracellular Ca2+ concentration. GO terms related to “antioxidant activity” and “superoxide dismutase activity” were significantly enriched in the two comparisons. The result of enzyme activity showed that the activities of superoxide dismutase and catalase in serum were significantly upregulated after activation of the cholinergic system but downregulated after blockade of this system. Furthermore, the cholinergic system maybe participate in regulating the “DNA replication”, “cell cycle”, “mismatch repair”, and “apoptosis” of the hemocytes after transplantation. These results provide new insights into the molecular mechanism of the cholinergic system in regulating the immunity of pearl oyster transplantation and facilitate the development of immunosuppressive agents to improve the effectiveness of cultured pearls.

An exploratory MALDI-TOF MS library based on SARAMIS superspectra for rapid identification of Aspergillus section Nigri.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a broadly used technique for identification and typing of microorganisms. However, its application to filamentous fungi has been delayed. The objective of this study was to establish a data library for rapid identification of the genus Aspergillus sect. Nigri using MALDI-TOF MS. With respect to sample preparation, we compared the utility of using mature mycelia, including conidial structures, to accumulate a wider range of proteins versus the conventional method relying on young hyphae. Mass spectral datasets obtained for 61 strains of 17 species were subjected to cluster analysis and compared with a phylogenetic tree based on calmodulin gene sequences. Specific and frequent mass spectral peaks corresponding to each phylogenetic group were selected (superspectra for the SARAMIS system). Fifteen superspectra representing nine species were ultimately created. The percentage of correct identification for 217 spectra was improved from 36.41% to 86.64% using the revised library. Additionally, 2.76% of the spectra were assigned to candidates that comprised several related species, including the correct species.

Molecular Phylogenetic Diversity and Biological Characterization of Diaporthe Species Associated with Leaf Spots of Camellia sinensis in Taiwan.

Camellia sinensis is one of the major crops grown in Taiwan and has been widely cultivated around the island. Tea leaves are prone to various fungal infections, and leaf spot is considered one of the major diseases in Taiwan tea fields. As part of a survey on fungal species causing leaf spots on tea leaves in Taiwan, 19 fungal strains morphologically similar to the genus Diaporthe were collected. ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α), tub2 (beta-tubulin), and cal (calmodulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. In total, six Diaporthe species, including one new species, Diaporthe hsinchuensis, were identified as linked with leaf spot of C. sinensis in Taiwan based on both phenotypic characters and phylogeny. These species were further characterized in terms of their pathogenicity, temperature, and pH requirements under laboratory conditions. Diaporthe tulliensis, D. passiflorae, and D. perseae were isolated from C. sinensis for the first time. Furthermore, pathogenicity tests revealed that, with wound inoculation, only D. hongkongensis was pathogenic on tea leaves. This investigation delivers the first assessment of Diaporthe taxa related to leaf spots on tea in Taiwan.

Seven New Records of Penicillium Species Belonging to Section Lanata-Divaricata in Korea

Penicillium species are known to be ubiquitous environmental saprophytes. In the survey of diversity of genus Penicillium, seven new records of Penicillium species belonging to section Lanata-Divaricata were isolated from freshwater and soil samples collected from different locations in Korea. Based on morphological characteristics and multilocus phylogenetic analysis of the rDNA internal transcribed spacer region (ITS), β-tubulin (BenA), and calmodulin (CaM) genes, the isolated strains were identified as P. annulatum, P. camponotum, P. echinulonalgiovense, P. globosum, P. limosum, P. onobense, and P. yunnanense, respectively. This study presents detailed phylogenetic analyses and morphological descriptions of these species that contribute to section Lanata-Divaricata in Korea.

Species diversity, novel interactions and absence of well-supported host-guided phylogenetic groupings of Neotropical Alternaria isolates causing foliar lesions in Solanaceae.

To report the characterization of 120 Alternaria isolates inducing early blight-like foliar lesions in nine species of five Solanaceae genera collected across all macrogeographical Brazilian regions. Phylogenetic relationships were assessed via analyses of the Alternaria alternata allergenic protein-coding, glyceraldehyde-3-phosphate dehydrogenase and the calmodulin gene sequences. Most of the tomato isolates were placed into the Alternaria linariae cluster, whereas most of the potato isolates were grouped with Alternaria grandis. Novel host-pathogen interactions were also reported. Seventeen isolates were selected for morphometrical characterization, and a subsample of 13 isolates was employed in pathogenicity assays on tomato, potato, eggplant, scarlet eggplant, Capsicum annuum, Datura stramonium, Physalis angulata and Nicotiana tabacum. Eleven isolates were able to induce foliar lesions in tomatoes but none in C. annuum. Potato was susceptible to a subgroup of isolates but displayed a subset of isolate-specific interactions. Morphological traits were in overall agreement with molecular and host range data. Alternaria linariae and A. grandis were confirmed as the major causal agents of tomato and potato early blight, respectively. However other Alternaria species are also involved with early blight in solanaceous hosts in Brazil. The diversity and host-specific patterns of the Alternaria isolates from Solanaceae may have practical implications in establishing effective early blight genetic resistance and cultural management strategies especially for tomato and potato crops.

Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025–0.041 and 0.211–0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020–0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA’s did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Molecular Diversity of Aspergilli in Two Iranian Hospitals

The Aspergillus species are main causative agents of various infections such as invasive aspergillosis (IA) in immunocompromised patients and these infections have high mortality rates. In this study, we provide insight in the species causing aspergillosis in Iran based on morphology and sequence data. Clinical (n = 117) and environmental isolates (n = 54) collected during 2010-2016 from University hospitals in Mashhad and Tehran (Iran) were identified both morphologically and molecularly using partial calmodulin (CaM) gene sequences. Clinical cases were identified based on EORTC/MSG criteria. Aspergillus flavus (n = 96, 55%) was the most prevalent species among the clinical and environmental isolates while A. fumigatus (n = 13, 7.5%) ranked fourth after A. tubingensis (n = 23, 13%) and A. welwitchiae (n = 18, 10%). Species such as A. tubingensis, A. welwitschiae, A. fumigatus, A. sydowii, A. neoniger and A. terreus were present in both clinical and environmental samples indicating the possible environmental source of infections. Interestingly, A. niger was isolated only once. Furthermore, 13 other rare and cryptic Aspergillus species were detected. Pulmonary and respiratory disorders (n = 33), followed by transplantation (n = 23), invasive fungal rhinosinusitis (n = 14), and haematological malignancies (n = 12) were major predisposing factors. According to EORTC/MSG criteria, there were 43 probable cases identified followed by 36 cases for each of proven and possible ones. Correct molecular identification will be useful for further epidemiological studies.

First Report of Anthracnose Caused by Colletotrichum siamense and C. fructicola of Camellia chrysantha in China.

Camellia chrysantha (Hu) Tuyama, belonging to the Theaceae family, is famous for its large size and golden yellow flowers, which has high ornamental and health care functions (Mo et al. 2013). Anthracnose is one of the most important fungal diseases worldwide, causing serious economic losses to many plants. In October 2019, severe anthracnose symptoms were observed on the leaves of C. chrysantha in a 0.6 hectare field with 15-20% disease incidence in Fangchenggang city, Guangxi Zhuang Autonomous Region of China. Diseased leaves initially appeared irregular chlorotic spots, which afterwards enlarged and coalesced. Finally, the spots became dark brown or black, sunken lesions (8-22 mm in diameter), and covered with plenty of acervuli. For pathogen isolation, the leaf lesions were cut into small tissue pieces (5 mm×5 mm), disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated at 28°C on potato dextrose agar (PDA) plates. A total of 7 fungal isolates with whitish to light grey, dense colonies were recovered at 5 days. These isolates were tentatively identified as belonging to Colletotrichum gloeosporioides species complex through morphological and cultural characters (Weir et al. 2012). The conidia were nonseptate, cylindrical with obtuse to rounded ends, 13.9 to 18.3 (average 16.1) μm × 4.5 to 6.2 (average 5.4) μm (n = 50). For further precise identification, the 7 Colletotrichum isolates were analyzed using partial sequences of genomic loci including the internal transcribed spacer (ITS), β-tubulin (TUB), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and the mating type locus MAT1-2 (ApMat) genes (Liu et al. 2015). The amplification sequences were compared with the sequences registered in the GenBank database based on nucleotide similarity. The above sequences of 4 isolates (JZB-PF4232, JZB-PF2231, JZB-PF42 and JZB-PF22) had 99-100% identity to the sequences of Colletotrichum siamense strains retrieved from GenBank, while the sequences of the other 3 isolates (JZB-PF3231, JZB-PF32 and JZB-PF41) showed over 99% identity with those of the C. fructicola strains. All the sequences were deposited in GenBank with accession number MT708987 to MT709007, MW149430 to MW149433, and MW142259 to MW142282. A multi-loci phylogenetic analysis of the concatenated sequences of ITS, TUB, CAL, ACT, GAPDH, GS and ApMat genes placed the 4 isolates described above in the C. siamense clade, while the other 3 isolates was attribute to the C. fructicola clade. Pathogenicity tests were conducted on 7 healthy 2-year-old C. chrysantha seedlings (cv. Fangpu), consisted of 21 wounded leaves made by a sterile needle, with 3 leaves per seedling. Artificial inoculations were performed by treating each seedling with 20 µl of spore suspension (106 conidia/ml) of each isolate. Leaves of seedlings treated with sterilized water under the same conditions served as controls. The experiment was repeated three times. All the seedlings were covered with plastic bags to maintain high humidity (90% RH) and placed in a greenhouse kept at 25°C with a 16 h light / 8 h dark photoperiod. After 8 days, the inoculated leaves of C. chrysantha plants developed typical dark brown or black lesions, similar to the symptoms in the field, whereas controls remained symptomless. Koch's postulates were fulfilled by re-isolation of the same fungi from symptomatic inoculated leaves, identification confirmed by morphological and molecular characteristics, respectively. C. siamense and C. fructicola have been found to cause anthracnose on Camellia sinensis (Wang et al. 2016; Shi et al. 2018). C. fructicola has also been reported to cause anthracnose on Citrus sinensis in China (Hu et al. 2019). To our knowledge, this is the first report of C. siamense and C. fructicola causing anthracnose on C. chrysantha in China.

Occurrence of Alternaria leaf blight of sunflower caused by two closely related species Alternaria solani and A. tomatophila in Inner Mongolia

Sunflower (Helianthus annuus L.) is an important oilseed crop in China and is grown in rotation with potato in the northern parts of China, including Inner Mongolia, Hebei, Shaanxi, Shanxi, Gansu, Jilin, Liaoning and Heilongjiang provinces. In 2014, diseased samples of sunflower (variety 3638C) with black sunken lesions on bracts and stems of sunflower were collected in Wuchuan, Hohhot (41°05′ N, 111°27′ E) and Chahaeryouyihouqi, Wulanchabu (41°52′ N, 113°0′ E), Inner Mongolia, China. We used morphological and molecular techniques to identify the causal agent of Alternaria leaf blight. Phylogenetic analysis based on a combined phylogenetic dataset using the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major allergen gene (Alt a1) was done to support the morphological identification. In addition, PCR was performed with specific primers targeted on Alt a1 and calmodulin genes of A. solani and A. tomatophila. The results showed that HaAs7 gave an amplicon of the expected size (164 bp) with A. solani-specific primers, however, no band was observed with the A. tomatophila-specific primers. The opposite pattern of PCR result (483 bp) was obtained with DNA of HaAt4 as template, indicating that HaAs7 is A. solani and HaAt4 is A. tomatophila. Furthermore, evaluation the pathogenicity of the recovered Alternaria isolates and their potential as causal agents of Alternaria leaf blight of sunflower was done. Pathogenicity tests showed that A. solani and A. tomatophila tested could be capable of causing Alternaria leaf blight of sunflower. Till now, nine species of Alternaria have been reported causing Alternaria leaf blight of sunflower, this is the first report that A. solani and A. tomatophila as causal pathogens for Alternaria leaf blight on sunflower.

Far-infrared irradiation inhibits breast cancer cell proliferation independently of DNA damage through increased nuclear Ca2+/calmodulin binding modulated-activation of checkpoint kinase 2

Far-infrared (FIR) irradiation is reported to inhibit cell proliferation in various types of cancer cells; the underlying mechanism, however, remains unclear. We explored the molecular mechanisms using MDA-MB-231 human breast cancer cells. FIR irradiation significantly inhibited cell proliferation and colony formation compared to hyperthermal stimulus, with no alteration in cell viability. No increase in DNA fragmentation or phosphorylation of DNA damage kinases including ataxia-telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase, and DNA-dependent protein kinase indicated no DNA damage. FIR irradiation increased the phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (p-Chk2-Thr68) but not that of checkpoint kinase 1 at Ser345. Increased nuclear p-Chk2-Thr68 and Ca2+/CaM accumulations were found in FIR-irradiated cells, as observed in confocal microscopic analyses and cell fractionation assays. In silico analysis predicted that Chk2 possesses a Ca2+/calmodulin (CaM) binding motif ahead of its kinase domain. Indeed, Chk2 physically interacted with CaM in the presence of Ca2+, with their binding markedly pronounced in FIR-irradiated cells. Pre-treatment with a Ca2+ chelator significantly reversed FIR irradiation-increased p-Chk2-Thr68 expression. In addition, a CaM antagonist or small interfering RNA-mediated knockdown of the CaM gene expression significantly attenuated FIR irradiation-increased p-Chk2-Thr68 expression. Finally, pre-treatment with a potent Chk2 inhibitor significantly reversed both FIR irradiation-stimulated p-Chk2-Thr68 expression and irradiation-repressed cell proliferation. In conclusion, our results demonstrate that FIR irradiation inhibited breast cancer cell proliferation, independently of DNA damage, by activating the Ca2+/CaM/Chk2 signaling pathway in the nucleus. These results demonstrate a novel Chk2 activation mechanism that functions irrespective of DNA damage.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin

BackgroundCystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs.MethodsProtoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively.ResultsAt 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 μM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 μM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations.ConclusionsTropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.Graphical

Identification and in vitro antifungal susceptibility of causative agents of onychomycosis due to Aspergillus species in Mashhad, Iran

Aspergillus species are emerging causative agents of non-dermatophyte mold onychomycosis. In this study, 48 Aspergillus isolates were obtained from patients with onychomycosis in Mashhad, Iran, during 2015–2018. The aim is to identify the Aspergillus isolates to the species level by using partial calmodulin and beta-tubulin gene sequencing and MALDI-TOF MS, and to evaluate their in vitro susceptibility to ten antifungal drugs: terbinafine, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin, anidulafungin and amphotericin B according to CLSI M38-A3. Our results indicate that A.flavus (n = 38, 79%) is the most common Aspergillus species causing onychomycosis in Mashhad, Iran. Other detected species were A. terreus (n = 3), A. tubingensis (n = 2), A. niger (n = 1), A. welwitschiae (n = 1), A. minisclerotigenes (n = 1), A. citrinoterreus (n = 1) and A. ochraceus (n = 1). Aspergillus flavus, A. terreus and A. niger isolates were correctly identified at the species level by MALDI-TOF MS, while all cryptic species were misidentified. In conclusion, A. flavus is the predominant Aspergillus species causing onychomycosis due to Aspergillus spp. in Mashhad, Iran. MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly for common Aspergillus species. It is important that the current database of reference spectra, representing different Aspergillus species is expanded to increase the precision of the species-level identification. Terbinafine, posaconazole and echinocandins were in vitro most active against the studies Aspergillus isolates and terbinafine could be the first choice for treatment of onychomycosis due to Aspergillus.

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China.

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) μm long × (13-21) μm wide (n=30) in V8 juice agar, (22-38) μm long × (11-19) μm wide (n=30) in PCA, (21-41) μm long × (13-20) μm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 μm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

Diversity of Diaporthe species associated with melanose disease on citrus trees in Jiangxi Province, China

Citrus melanose is an important disease that seriously damages citrus production. It is caused by Diaporthe species, which are known as important pathogens, endophytes or saprobes with broad host ranges. Over 170 species of Diaporthe were reported around the world and the complexity of species structures makes it hard to be identified. Phylogenetic analysis based on translation elongation factor 1-ɑ (TEF1) gene, s-tubulin (TUB) gene, calmodulin (CaM) gene, histone (HIS) gene and ITS locus was a reliable method to identify Diaporthe species In this study, the species structure of Diaporthe species from different tissues within individual trees was investigated. Altogether, 494 single spore isolates were obtained, and 92 of them were selected to sequence the five loci (ITS, TEF1, HIS, TUB, CaM) for phylogenetic analysis. Based on the combined multi-locus dataset, phylogenetic trees constructed by using two methods (MP and ML) showed that D. citri was the dominant species no matter on fruits, leaves or twigs. Diaporthe species showed the greatest diversity on fruits and the least diversity on twigs. D. eres was only isolated from twigs. The results of this study may enhance our understanding for the species diversity of Diaporthe species on different tissues from same trees, such information would be useful for the control of Diaporthe diseases in the future.

Development and application of Recombinase Polymerase Amplification assay for detection of Bipolaris sorokiniana

Bipolaris sorokiniana is a fungal pathogen that caused root rot and spot blotch on wheat. A Recombinase Polymerase Amplification (RPA) assay was developed to detect B. sorokiniana specifically based on the sequences of calmodulin gene sequence. The specificity of RPA assay was tested against 19 fungal organisms associated with wheat, and the detection limit was determined to be 10 pg for B. sorokiniana pure fungal DNA. Further, the RPA assay was capable of detecting B. sorokiniana from artificially inoculated and field collected wheat tissues directly. These results indicate that the RPA assay is rapid and effective method for detection and molecular identification of B. sorokiniana.

Identification and expression analysis of a calmodulin gene in Phycocalida chauhanii (Rhodophyceae, Bangiales)

Calmodulin (CaM) plays an important regulatory role in plant growth and development. In this study, on the basis of previous transcriptomic annotation a homolog of CaM gene (Pc-CaM7) was identified from the red alga Phycocalida chauhanii (previously known as Porphyra chauhanii). It encoded a 151 amino acids protein with 4 EF-hand regions, which was the typical structure of calmodulin family proteins. Evolutionary analysis showed that Pc-CaM7 shared conserved orthologous relationships with CaMs from Neoporphyra haitanensis (formerly Pyropia haitanensis), Porphyra umbilicalis, and Gracilariopsis chorda. During the developmental period of P. chauhanii gametophytic germlings from 25- to 50-day-old, the asexual archeospores were continuedly released from the marginal region of blades, followed by the emergence of spermatangia from 40-day-old germlings. Based on quantitative real-time PCR analysis, it showed that expression trend of Pc-CaM7 was correlated with the rule of archeospores’ formation and release, whereas in N. haitanensis without archeospore characteristic, the expression pattern of the orthologous gene (Pha_00404) was different, which increased and reached a plateau during the same developmental period. These findings suggested that Pc-CaM7 may be involved in the process of archeospores formation and release, which may provide a foundation for understanding the molecular mechanisms of asexual reproduction in Bangiales.

Phylogenetic and Haplotype Network Analyses of Diaporthe eres Species in China Based on Sequences of Multiple Loci.

Diaporthe eres is considered one of the most important causal agents of many plant diseases, with a broad host range worldwide. In this study, multiple sequences of ribosomal internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF1-α), beta-tubulin gene (TUB2), calmodulin gene (CAL), and histone-3 gene (HIS) were used for multi-locus phylogenetic analysis. For phylogenetic analysis, maximum likelihood (ML), maximum parsimony (MP), and Bayesian inferred (BI) approaches were performed to investigate relationships of D. eres with closely related species. The results strongly support that the D. eres species falls into a monophyletic lineage, with the characteristics of a species complex. Phylogenetic informativeness (PI) analysis showed that clear boundaries could be proposed by using EF1-α, whereas ITS showed an ineffective reconstruction and, thus, was unsuitable for speciating boundaries for Diaporthe species. A combined dataset of EF1-α, CAL, TUB2, and HIS showed strong resolution for Diaporthe species, providing insights for the D. eres complex. Accordingly, besides D. biguttusis, D. camptothecicola, D. castaneae-mollissimae, D. cotoneastri, D. ellipicola, D. longicicola, D. mahothocarpus, D. momicola, D. nobilis, and Phomopsis fukushii, which have already been previously considered the synonymous species of D. eres, another three species, D. henanensis, D. lonicerae and D. rosicola, were further revealed to be synonyms of D. eres in this study. In order to demonstrate the genetic diversity of D. eres species in China, 138 D. eres isolates were randomly selected from previous studies in 16 provinces. These isolates were obtained from different major plant species from 2006 to 2020. The genetic distance was estimated with phylogenetic analysis and haplotype networks, and it was revealed that two major haplotypes existed in the Chinese populations of D. eres. The haplotype networks were widely dispersed and not uniquely correlated to specific populations. Overall, our analyses evaluated the phylogenetic identification for D. eres species and demonstrated the population diversity of D. eres in China.

Endophytic Diaporthe Associated With Citrus grandis cv. Tomentosa in China.

Diaporthe species are associated with Citrus as endophytes, pathogens, and saprobes worldwide. However, little is known about Diaporthe as endophytes in Citrus grandis in China. In this study, 24 endophytic Diaporthe isolates were obtained from cultivated C. grandis cv. “Tomentosa” in Huazhou, Guangdong Province in 2019. The nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (tef1), β-tubulin (tub2), and partial calmodulin (cal) gene regions were sequenced and employed to construct phylogenetic trees. Based on morphology and combined multigene phylogeny, eleven Diaporthe species were identified including two new species, Diaporthe endocitricola and D. guangdongensis. These are the first report of D. apiculata, D. aquatica, D. arecae, D. biconispora, D. limonicola, D. masirevicii, D. passifloricola, D. perseae, and D. sennae on C. grandis. This study provides the first intensive study of endophytic Diaporthe species on C. grandis cv. tomentosa in China. These results will improve the current knowledge of Diaporthe species associated with C. grandis. The results obtained in this study will also help to understand the potential pathogens and biocontrol agents and to develop a platform in disease management.

First Report of Diaporthe hongkongensis Causing Fruit rot on Peach (Prunus persica) in China.

Peach [Prunus persica (L.) Batsch] is an important deciduous fruit tree in the family Rosaceae and is a widely grown fruit in China (Verde et al., 2013). In July and August 2018, a fruit rot disease was observed in a few peach orchards in Zhuzhou city, the Hunan Province of China. Approximately 30% of the fruit in more than 400 trees was affected. Symptoms displayed were brown necrotic spots that expanded, coalesced, and lead to fruit being rotten. Symptomatic tissues excised from the margins of lesions were surface sterilized in 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and incubated on potato dextrose agar (PDA) at 26°C in the dark. Fungal colonies with similar morphology developed, and eight fungal colonies were isolated for further identification. Colonies grown on PDA were grayish-white with white aerial mycelium. After an incubation period of approximately 3 weeks, pycnidia developed and produced α-conidia and β-conidia. The α-conidia were one-celled, hyaline, fusiform, and ranged in size from 6.0 to 8.4 × 2.1 to 3.1 μm, whereas the β-conidia were filiform, hamate, and 15.0 to 27.0 × 0.8 to 1.6 μm. For molecular identification, total genomic DNA was extracted from the mycelium of a representative isolate HT-1 and the internal transcribed spacer region (ITS), β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), calmodulin (CAL), and histone H3 gene (HIS) were amplified and sequenced (Meng et al. 2018). The ITS, TUB, TEF1, CAL and HIS sequences (GenBank accession nos. MT740484, MT749776, MT749778, MT749777, and MT749779, respectively) were obtained and in analysis by BLAST against sequences in NCBI GenBank, showed 99.37 to 100% identity with D. hongkongensis or D. lithocarpus (the synonym of D. hongkongensis) (Gao et al., 2016) (GenBank accession nos. MG832540.1 for ITS, LT601561.1 for TUB, KJ490551.1 for HIS, KY433566.1 for TEF1, and MK442962.1 for CAL). Pathogenicity tests were performed on peach fruits by inoculation of mycelial plugs and conidial suspensions. In one set, 0.5 mm diameter mycelial discs, which were obtained from an actively growing representative isolate of the fungus on PDA, were placed individually on the surface of each fruit. Sterile agar plugs were used as controls. In another set, each of the fruits was inoculated by application of 1 ml conidial suspension (105 conidia/ml) by a spray bottle. Control assays were carried out with sterile distilled water. All treatments were maintained in humid chambers at 26°C with a 12-h photoperiod. The inoculation tests were conducted twice, with each one having three fruits as replications. Six days post-inoculation, symptoms of fruit rot were observed on inoculated fruits, whereas no symptoms developed on fruits treated with agar plugs and sterile water. The fungus was re-isolated and identified to be D. hongkongensis by morphological and molecular methods, thus fulfilling Koch's Postulates. This fungus has been reported to cause fruit rot on kiwifruit (Li et al. 2016) and is also known to cause peach tree dieback in China (Dissanayake et al. 2017). However, to our knowledge, this is the first report of D. hongkongensis causing peach fruit rot disease in China. The identification of the pathogen will provide important information for growers to manage this disease.

Mycoflora and DNA sequencing analysis of radioresistant fungi isolated from irradiated vehicular air conditioning filters collected in São Paulo, Brazil

Fungal bioburdens in filters from car air conditioning systems represent a potential risk of respiratory or pulmonary diseases to both passengers and drivers. The present study demonstrated high fungal contamination in air conditioning filters collected from fifty-seven vehicles from the city of São Paulo, Brazil. The filters samples were irradiated with 10, 15 and 20 kGy gamma ray doses with a cobalt-60 source. The presence of Aspergillus flavus, Penicillium glabrum and Fusarium incarnatum-equiseti remaining after the ionizing treatment with 10 and 15 kGy. The radioresistant pathogenic species was confirmed through genetic sequencing of the ITS, β-tubulin and calmodulin gene rDNA regions. The 20 kGy dose was efficient in inhibiting pathogenic fungi growth in all samples but promoted the fungal decontamination in 79% of the samples. These results and other efforts will enable ionizing radiation to become an important tool in contributing to the recycling of automotive vehicle air filters and in ensuring indoor air quality for both drivers and passengers. Keywords: Fungal contamination, indoor air quality, vehicle air filters, ionizing radiation.