Medication-related osteonecrosis of the jaw (MRONJ): a review of pathogenesis hypothesis and therapy strategies.

Medication-related osteonecrosis of the jaw (MRONJ), a severe side effect caused by antiresorptive antiangiogenic medication, particularly bisphosphonates (BPs), has become a challenging disease with serious and profound effects on the physical and mental health of patients. Although it occurs with high frequency and is harmful, the exact mechanism of MRONJ remains unknown, and systematic and targeted approaches are still lacking. Maxillofacial surgeons focus on the etiology of osteonecrosis in the mandible and maxilla as well as the appropriate oral interventions for high-risk patients. Adequate nursing care and pharmacotherapy management are also crucial. This review provides a current overview of the clinicopathologic feature and research of MRONJ caused by BPs, with an emphasis on the potential mechanisms and current therapy and prevention strategies of the disease. We are of the opinion that an in-depth comprehension of the mechanisms underlying MRONJ will facilitate the development of more precise and efficacious therapeutic approaches, resulting in enhanced clinical outcomes for patients.

First report of Black Spot on Eucalyptus grandis × Eucalyptus urophylla caused by Pseudoplagiostoma eucalypti in Guangxi, China.

Eucalyptus grandis × Eucalyptus urophylla hybrid clone is an economically and ecologically important forest variety and is widely planted in Guangxi, China. Black spot, a newly found disease, occurred nearly 5333.3 hectares in an E. grandis × E. urophylla plantation of Qinlian forest farm (N: 21.866°, E: 108.921°) in Guangxi in October, 2019. Infected plants had lesions of black spots with watery margins on petioles and veins of E. grandis × E. urophylla. The size of spots ranged between 3 to 5 mm in diameter. When lesions expanded to girdle the petioles, wilt and death of leaves was observed, which subsequently affected growth of the trees. To isolate the causal agent, symptomatic plant tissues (leaves and petioles) were collected from two different sites, sampled from five plants each site. In the lab, infected tissues were surface sterilized with 75% ethanol for 10 seconds, then 2% sodium hypochlorite for 120 seconds, and rinsed with sterile distilled water three times. Small segments (5×5 mm) were cut from the margins of the lesions, then placed on potato dextrose agar (PDA) plates. The plates were incubated at 26°C in dark for 7 to 10 days. Fungal isolates YJ1 and YM6 with a similar morphology, which were obtained from 14 of 60 petioles and 19 of 60 veins respectively. These two colonies were initially light orange, then turned to olive brown as time progressed. Conidia were hyaline, smooth, aseptate, ellipsoidal, apex obtuse, and base tapering to flat protruding scar, 16.8 to 26.5µm long, and 6.6 to 10.4 µm wide (n=50). Some conidia had one or two guttules. The morphological characteristics were consistent with the description of Pseudoplagiostoma eucalypti Cheew., M. J. Wingf. & Crous (Cheewangkoon et al. 2010). For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 and T1/Bt2b, respectively (White et al. 1990; O'Donnell et al.1998; Glass and Donaldson 1995). Sequences of the two strains were deposited in GenBank (ITS: MT801070 and MT801071; BT2: MT829072 and MT829073). Phylogenetic tree was constructed with a maximum likelihood method, revealing that YJ1 and YM6 were on the same branch with P. eucalypti. Pathogenicity tests of the two strains were performed on three-month-old E. grandis × E. urophylla seedlings, by inoculating 6 wounded (by stabbing on petioles or veins) leaves of seedlings with mycelial PDA plugs (5 ×5 mm) from the edge of a 10-day old colony of strain YJ1 or YM6. Another 6 leaves were treated in the same manner but with PDA plugs as controls. All treatments were incubated in humidity chambers at 27°C and 80% relative humidity, under ambient light. All experiments were conducted three times. Lesions were observed at the points of inoculation, the petioles or veins turned black on inoculated leaves after 7 days, wilting of the leaves were also observed after 30 days, however the controls remained asymptomatic. Re-isolation was made and the fungus had same morphological measurements as the inoculated fungus, thus completing Koch's postulates. P. eucalypti had been reported as a pathogen of leaf spot on E. robusta in Taiwan island (Wang et al. 2016), leaf and shoot blight on E. pulverulenta in Japan (Inuma et al. 2015). To our knowledge, this is the first report of P. eucalypti affecting E. grandis × E. urophylla in mainland China. This report provides basis for the rational prevention and control of this new disease in the cultivation process of E. grandis × E. urophylla.

Streptococcus mutans sigX-inducing peptide inhibits the virulence of Candida albicans and oral candidiasis through the Ras1-cAMP-Efg1 pathway.

Oral candidiasis is the most common fungal infectious disease in the human oral cavity, and Candida albicans is the major pathogenic agent. Increasing drug resistance and the lack of new types of antifungals greatly increase the challenges for treating fungal infections. Targeting hyphal transition provides a promising strategy to inhibit the virulence of C. albicans and overcome drug resistance. This study aimed to investigate the effects and mechanisms of sigX-inducing peptide (XIP), a quorum-sensing signal peptide secreted by Streptococcus mutans, on C. albicans hyphal development and biofilm formation in vitro and oropharyngeal candidiasis in vivo. XIP significantly inhibited C. albicans yeast-to-hypha transition and biofilm formation in a dose-dependent manner from 0.01 to 0.1 µM. XIP significantly downregulated expression of genes from the Ras1-cAMP-Efg1 pathway (RAS1, CYR1, TPK2, EFG1 and UME6), a key pathway to regulate C. albicans hyphal development. Importantly, XIP reduced the levels of key molecules cAMP and ATP from this pathway, while the addition of exogenous cAMP and overexpression of RAS1 restored the hyphal development inhibited by XIP. XIP also lost its hyphal inhibitory effects on ras1Δ/Δ and efg1Δ/Δ strains. These results further confirmed that XIP inhibited hyphal development through downregulation of the Ras1-cAMP-Efg1 pathway. A murine oropharyngeal candidiasis model was employed to evaluate the therapeutic effects of XIP on oral candidiasis. XIP effectively reduced the infected epithelial area, fungal burden, hyphal invasion and inflammatory infiltrates. These results revealed the antifungal effects of XIP, and highlighted that XIP can be a potential antifungal peptide against C. albicans infection.

First Report of Leaf Spot Caused by Lasiodiplodia theobromae on Kadsura coccinea in China.

Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 µm (average 26.07×14.51 µm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and ß-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 µL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 µL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Illicium difengpi in China.

Illicium difengpi B. N. Chang et al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initial leaf symptoms were round spots with gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomatic leaves were collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affected leaves. Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n = 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n = 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 for ITS, OL539429 for ACT, OL539430 for tef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the corresponding ITS (MT649668), ACT (MN958710), tef1-α (MN958711) and GPDH (KU716077) sequences of P. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACT by the maximum likelihood method showed that isolate GX-2 clustered together with P. capitalensis. The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 µL spore suspension (1 × 106 spores/ml). Another four sterilized leaves were inoculated with 20 µL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy. P. capitalensis was successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensis caused leaf spots on various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report of P. capitalensis causing leaf spot on I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology of this disease should be investigated in further research.

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis.

Mining novel specific molecular targets and establishing efficient identification methods are significant for detecting Pseudomonas aeruginosa, which can enable P. aeruginosa tracing in food and water. Pangenome analysis was used to analyze the whole genomic sequences of 2017 strains (including 1,000 P. aeruginosa strains and 1,017 other common foodborne pathogen strains) downloaded from gene databases to obtain novel species-specific genes, yielding a total of 11 such genes. Four novel target genes, UCBPP-PA14_00095, UCBPP-PA14_03237, UCBPP-PA14_04976, and UCBPP-PA14_03627, were selected for use, which had 100% coverage in the target strain and were not present in nontarget bacteria. PCR primers (PA1, PA2, PA3, and PA4) and qPCR primers (PA12, PA13, PA14, and PA15) were designed based on these target genes to establish detection methods. For the PCR primer set, the minimum detection limit for DNA was 65.4 fg/µl, which was observed for primer set PA2 of the UCBPP-PA14_03237 gene. The detection limit in pure culture without pre-enrichment was 105 colony-forming units (CFU)/ml for primer set PA1, 103 CFU/ml for primer set PA2, and 104 CFU/ml for primer set PA3 and primer set PA4. Then, qPCR standard curves were established based on the novel species-specific targets. The standard curves showed perfect linear correlations, with R 2 values of 0.9901 for primer set PA12, 0.9915 for primer set PA13, 0.9924 for primer set PA14, and 0.9935 for primer set PA15. The minimum detection limit of the real-time PCR (qPCR) assay was 102 CFU/ml for pure cultures of P. aeruginosa. Compared with the endpoint PCR and traditional culture methods, the qPCR assay was more sensitive by one or two orders of magnitude. The feasibility of these methods was satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 29 ready-to-eat vegetable samples and was almost consistent with that of the national standard detection method. The developed assays can be applied for rapid screening and detection of pathogenic P. aeruginosa, providing accurate results to inform effective monitoring measures in order to improve microbiological safety.

Complete genome sequence of biocontrol strain Paenibacillus peoriae HJ-2 and further analysis of its biocontrol mechanism.

BACKGROUND: Paris polyphylla is a herb widely used in traditional Chinese medicine to treat various diseases. Stem rot diseases seriously affected the yield of P. polyphylla in subtropical areas of China. Therefore, cost-effective, chemical-free, eco-friendly strategies to control stem rot on P. polyphylla are valuable and urgently needed. RESULTS: In this paper, we reported the biocontrol efficiency of Paenibacillus peoriae HJ-2 and its complete genome sequence. Strain HJ-2 could serve as a potential biocontrol agent against stem rot on P. polyphylla in the greenhouse and field. The genome of HJ-2 consists of a single 6,001,192 bp chromosome with an average GC content of 45% and 5,237 predicted protein coding genes, 39 rRNAs and 108 tRNAs. The phylogenetic tree indicated that HJ-2 is most closely related to P. peoriae IBSD35. Functional analysis of genome revealed numerous genes/gene clusters involved in plant colonization, biofilm formation, plant growth promotion, antibiotic and resistance inducers synthesis. Moreover, metabolic pathways that potentially contribute to biocontrol mechanisms were identified. CONCLUSIONS: This study revealed that P. peoriae HJ-2 could serve as a potential BCA against stem rot on P. polyphylla. Based on genome analysis, the genome of HJ-2 contains more than 70 genes and 12 putative gene clusters related to secondary metabolites, which have previously been described as being involved in chemotaxis motility, biofilm formation, growth promotion, antifungal activity and resistance inducers biosynthesis. Compared with other strains, variation in the genes/gene clusters may lead to different antimicrobial spectra and biocontrol efficacies.

First Report of Anthracnose on Kadsura coccinea Caused by Colletotrichum siamense in China.

Kadsura coccinea (Lem.) A. C. Smith, an evergreen liana, is widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown numerous biological activities, such as anti-tumor, anti-HIV, and anti-oxidant (Yang et al. 2020). In June 2019, an anthracnose on K. coccinea was observed in a plantation in Longan (23°03´N, 107°54´E), Guangxi province. Disease incidence was up to 30% in a plantation. Its symptoms began as small brown spots that expanded into nearly circular spots (Fig. 1A). To isolate pathogen, diseased leaves were collected. The leaves were sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 90 s, then rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed into potato dextrose agar (PDA) plates. The plates were incubated in an incubator at 25°C in dark for 2-3 days. Fungal colonies with similar morphology of 27 isolates were isolated from the 30 infected tissues. Six representative isolates (YB1 to YB6) were selected to further study their characterization. Fungal colonies were grayish-white, orange-yellow conidial masses could be observed in colonies (Fig. 1C). The mature conidia were colorless and transparent, elliptical, and single-celled, 13.0-21.0 × 4.0-8.0 µm (average 16.92 × 5.92 µm; n =100) (Fig. 1B). The DNA sequences of ribosomal internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1) and ß-tubulin (TUB2) were amplified by PCR using the primer pairs ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b (Wang et al. 2020), respectively. Sequences were submitted to GenBank (Accession nos. MZ040489 to MZ040494 for ITS, MZ069043 to MZ069048 for GAPDH, MZ069049 to MZ069054 for CAL, MZ069055 to MZ069060 for ACT, MZ069061 to MZ069066 for CHS-1, and MZ069067 to MZ069072 for TUB2). These sequences were 98%-100% identical to that of reference isolates JX010278, JX010019, JX009709, GQ856775, GQ856730, and JX010410 of Colletotrichum siamense CBS 125378 ex-type recorded in GenBank. Phylogenetic analysis of combined ITS, GAPDH, CAL, ACT, CHS-1, TUB2 genes with 16 sequences obtained from GenBank using maximum likelihood method showed that the six isolates clustered with two reference isolates of Colletotrichum siamense as a distinct clade (Fig. 2). Based on morphological characteristics and phylogenetic analysis, six isolates were identified as C. siamense. Pathogenicity tests were performed on young, fully expanded leaves of 1-year seedlings. Every leaf was punctured at 6 points on the right half and 6 points on the left half using a sterile needle. A 10 µl conidial suspension (1×106 conidia/ml) was inoculated on each wound on the left-half leaf and a 10 µl sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 2 days and after removing the bags, plants were maintained in a growth chamber at 28°C, 80% relative humidity, and a 12-h photoperiod. Anthracnose spots formed 2 to 3 days after inoculation, whereas the control leaves remained symptomless. Morphological characters matched the descriptions of C. siamense. The pathogen was previously reported to cause anthracnose on Aloe vera (Azad et al. 2020), postharvest anthracnose in mango (Liu et al. 2017), pod rot in cacao (Serrato-Diaz et al. 2020). To our knowledge, this is the first report of anthracnose on K. coccinea caused by C. siamense in China.

Genome duplication improves rice root resistance to salt stress.

BACKGROUND: Salinity is a stressful environmental factor that limits the productivity of crop plants, and roots form the major interface between plants and various abiotic stresses. Rice is a salt-sensitive crop and its polyploid shows advantages in terms of stress resistance. The objective of this study was to investigate the effects of genome duplication on rice root resistance to salt stress. RESULTS: Both diploid rice (HN2026-2x and Nipponbare-2x) and their corresponding tetraploid rice (HN2026-4x and Nipponbare-4x) were cultured in half-strength Murashige and Skoog medium with 150 mM NaCl for 3 and 5 days. Accumulations of proline, soluble sugar, malondialdehyde (MDA), Na(+) content, H(+) (proton) flux at root tips, and the microstructure and ultrastructure in rice roots were examined. We found that tetraploid rice showed less root growth inhibition, accumulated higher proline content and lower MDA content, and exhibited a higher frequency of normal epidermal cells than diploid rice. In addition, a protective gap appeared between the cortex and pericycle cells in tetraploid rice. Next, ultrastructural analysis showed that genome duplication improved membrane, organelle, and nuclei stability. Furthermore, Na(+) in tetraploid rice roots significantly decreased while root tip H(+) efflux in tetraploid rice significantly increased. CONCLUSIONS: Our results suggest that genome duplication improves root resistance to salt stress, and that enhanced proton transport to the root surface may play a role in reducing Na(+) entrance into the roots.

Genome duplication effects on pollen development and the interrelated physiological substances in tetraploid rice with polyploid meiosis stability.

The breeding of polyploid rice made no breakthrough for a long time because of low seed set. The discovery and application of polyploid meiosis stability (PMeS) material played a pivotal role in solving this problem. Our results indicated that genome duplication led to different outcomes in different rice cultivars in terms of pollen fertility, viability, and the accumulation of important physiological substances such as free proline and endogenous hormones. Pollen from the PMeS HN2026-4X lines showed a high fertility and viability similar to those of HN2026-2X (4X indicates tetraploid while 2X indicates the diploid), whereas both rates decreased dramatically in Balilla-4X. The results of pollen microstructure and ultrastructure investigations suggested that the pollen development pattern in HN2026-4X appeared normal at all stages, but a lot of changes were discovered in Balilla-4X. Stable meiosis, timely tapetum degradation, and normal mitochondria development were critical factors insuring the high frequency pollen fertility of PMeS rice. The free proline content increased markedly in HN2026-4X as compared to HN2026-2X, but it was decreased for Balilla-4X. Genome duplication effects on regulating endogenous hormones accumulation in pollen were evident, resulting in the clear difference between PMeS HN2026-4X and Balilla-4X. The accumulation of IAA, ZR, and GA in mature pollen distinguished HN2026-4X from Balilla-4X, which was linked to normal pollen development. In particular, the excessive accumulation of ABA at the meiosis stage may be correlated to disorganized meiosis in Balilla-4X. All the results provided unequivocal evidence that genome duplication played specific roles in the normal pollen development of PMeS HN2026-4X.