Species of Botryosphaeriaceae associated with citrus branch diseases in China.

Citrus is an important and widely cultivated fruit crop in South China. Although the species of fungal diseases of leaves and fruits have been extensively studied, the causal organisms of branch diseases remain poorly known in China. Species of Botryosphaeriaceae are known as important fungal pathogens causing branch diseases on citrus in the USA and Europe. To determine the diversity of Botryosphaeriaceae species associated with citrus branch diseases in China, surveys were conducted in the major citrus-producing areas from 2017 to 2020. Diseased tissues were collected from twigs, branches and trunks with a range of symptoms including cankers, cracking, dieback and gummosis. Based on morphological characteristics and phylogenetic comparison of the DNA sequences of the internal transcribed spacer region (ITS), the translation elongation factor 1-alpha gene (tef1), the ß-tubulin gene (tub2) and the DNA-directed RNA polymerase II second largest subunit (rpb2), 111 isolates from nine provinces were identified as 18 species of Botryosphaeriaceae, including Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa, Neofusicoccum parvum, and six previously undescribed species, namely Do. citrimurcotticola, L. guilinensis, L. huangyanensis, L. linhaiensis, L. ponkanicola and Sphaeropsis linhaiensis spp. nov. Botryosphaeria dothidea (28.8 %) was the most abundant species, followed by L. pseudotheobromae (23.4 %), which was the most widely distributed species on citrus, occurring in six of the nine provinces sampled. Pathogenicity tests indicated that all 18 species of Botryosphaeriaceae obtained from diseased citrus tissues in this study were pathogenic to the tested Citrus reticulata shoots in vitro, while not all species are pathogenic to the tested Cocktail grapefruit (C. paradisi × C. reticulata) shoots in vivo. In addition, Lasiodiplodia was the most aggressive genus both in vitro and in vivo. This is the first study to identify Botryosphaeriaceae species related to citrus branch diseases in China and the results provide a theoretical basis for the implementation of prevention and control measures. Citation: Xiao XE, Wang W, Crous PW, et al. 2021. Species of Botryosphaeriaceae associated with citrus branch diseases in China. Persoonia 47: 106-135. https://doi.org/10.3767/persoonia.2021.47.03.

Species of Botryosphaeriaceae associated with citrus branch diseases in China.

Citrus is an important and widely cultivated fruit crop in South China. Although the species of fungal diseases of leaves and fruits have been extensively studied, the causal organisms of branch diseases remain poorly known in China. Species of Botryosphaeriaceae are known as important fungal pathogens causing branch diseases on citrus in the USA and Europe. To determine the diversity of Botryosphaeriaceae species associated with citrus branch diseases in China, surveys were conducted in the major citrus-producing areas from 2017 to 2020. Diseased tissues were collected from twigs, branches and trunks with a range of symptoms including cankers, cracking, dieback and gummosis. Based on morphological characteristics and phylogenetic comparison of the DNA sequences of the internal transcribed spacer region (ITS), the translation elongation factor 1-alpha gene (tef1), the ß-tubulin gene (tub2) and the DNA-directed RNA polymerase II second largest subunit (rpb2), 111 isolates from nine provinces were identified as 18 species of Botryosphaeriaceae, including Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa, Neofusicoccum parvum, and six previously undescribed species, namely Do. citrimurcotticola, L. guilinensis, L. huangyanensis, L. linhaiensis, L. ponkanicola and Sphaeropsis linhaiensis spp. nov. Botryosphaeria dothidea (28.8 %) was the most abundant species, followed by L. pseudotheobromae (23.4 %), which was the most widely distributed species on citrus, occurring in six of the nine provinces sampled. Pathogenicity tests indicated that all 18 species of Botryosphaeriaceae obtained from diseased citrus tissues in this study were pathogenic to the tested Citrus reticulata shoots in vitro, while not all species are pathogenic to the tested Cocktail grapefruit (C. paradisi × C. reticulata) shoots in vivo. In addition, Lasiodiplodia was the most aggressive genus both in vitro and in vivo. This is the first study to identify Botryosphaeriaceae species related to citrus branch diseases in China and the results provide a theoretical basis for the implementation of prevention and control measures. Citation: Xiao XE, Wang W, Crous PW, et al. 2021. Species of Botryosphaeriaceae associated with citrus branch diseases in China. Persoonia 47: 106-135. https://doi.org/10.3767/persoonia.2021.47.03.

Two sphingomyelin synthase homologues regulate body weight and sphingomyelin synthesis in female brown planthopper, N. lugens (Stål).

Although sphingomyelins known to be are lipid constituents of the plasma membrane in vertebrates, much remains obscure about the metabolism of sphingomyelins in insects. With ultra performance liquid chromatography-time-of-flight-tandem mass spectrometry analysis, we revealed for the first time that sphingomyelins are abundant in Nilaparvata lugens (Stål), the brown planthopper (BPH), and their biosynthesis is carried out by sphingomyelin synthase-like protein 2 (SMSL2), which is homologous to sphingomyelin synthase-related protein (SMSr). Unlike other insect species, high concentrations of sphingomyelins rather than ceramide phosphoethanolamines exist in the BPH. Two putative genes, which are homologous to SMSr, are named Nilaparvata lugens SMS-like 1 (NlSMSL1) and 2 (NlSMSL2). Knockdowns of both NlSMSL2 and NlSMSL1 were conducted but only the first decreased concentrations of sphingomyelins in the BPH, indicating that NlSMSL2 plays a role in the biosynthesis of sphingomyelins. Real-time quantitative PCR analysis revealed both NlSMSL1 and NlSMSL2 are highly expressed in BPH adults, with NlSMSL1 specifically highly expressed in reproductive organs (ovaries and testes) whereas NlSMSL2 was highly expressed in the malpighian tubules. The knockdown of NlSMSL1 or NlSMSL2 increased BPH female body weight but not that of males, suggesting sex-specific roles for SMSLs in influencing BPH body weight. The results suggest that NlSMSL2 catalyses the synthesis of sphingomyelins and maintains female BPH body weight through alteration of sphingolipid content.

Identification and characterization of Laodelphax striatellus (Insecta: Hemiptera: Delphacidae) neutral sphingomyelinase.

The neutral sphingomyelinase (nSMase) 1 homologue gene LsSMase was cloned from Laodelphax striatellus, a direct sap-sucker and virus vector of gramineous plants, and expressed via a Bac to Bac baculovirus expression system. The LsSMase-enhanced green fluorescent protein fusion protein was located in the endoplasmic reticulum in a similar manner to mammalian nSMase 1. The biochemical properties of LsSMase were determined in detail. The optimal pH and temperature for recombinant LsSMase were 8 and 37 °C, respectively. LsSMase was an Mg2+ or Mn2+ dependent enzyme, but different concentration of each were needed. The activity of LsSMase was significantly stimulated by Ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), whereas it was inhibited by ethylenediaminetetraacetic acid. Millimolar concentrations of Zn2+ completely inhibited LsSMase. The reducing agents dithiothreitol and ß-mercaptoethanol varied in their effects on activity. Phospholipids were not found to stimulate LsSMase.

Identification and biochemical characterization of Laodelphax striatellus neutral ceramidase.

Ceramidases are a group of enzymes that catalyse hydrolysis of ceramides to generate fatty acid and sphingosine. In this study, we report the cloning and characterization of the rice small brown planthopper Laodelphax striatellus neutral ceramidase (nCDase), LsnCer. LsnCer was identified by sequencing the transcriptome of L. striatellus and is a protein of 717 amino acids with a predicted molecular weight of 79.3 kDa. Similarly to other known nCDases, the optimum pH for LsnCer is 8.0 and the optimum temperature is 37 °C for its in vitro activity. LsnCer activity is inhibited by Zn(2+) significantly and Fe(2+) slightly. LsnCer has broad substrate specificity with a preference for ceramides with a medium acyl-chain or a monounsaturated long acyl-chain. Infection with rice strip virus (RSV) or treatment with insecticides significantly increased LsnCer mRNA expression and its enzymatic activity in L. striatellus. These results suggest that LsnCer is a bona fide nCDase that may have a role in adaption of L. striatellus to environmental stresses.

Cloning, expression and functional analysis of a general odorant-binding protein 2 gene of the rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae).

A full-length cDNA encoding a general odorant binding protein 2 (GOBP2) was cloned from the antennae of the rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), by the combination of reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR). The cDNA contains a 489 bp open reading frame, which encodes a 162 amino acid protein, termed as Ch. suppressalis GOBP2 (CsupGOBP2). CsupGOBP2 is similar in the number of amino acids and protein sequence to GOBP2s in other species of Lepidoptera. RT-PCR results showed that CsupGOBP2 mRNA was highly expressed in the adult antennae of both females and males, as was CsupGOBP2 protein as revealed by Western blot analysis. CsupGOBP2 expressed in Escherichia coli was purified by affinity chromatography, refolding and gel filtration from the inclusion body. Fluorescence emission spectra and competitive binding assays by using N-phenyl-1-naphthylamine as first binding ligand and odorants as potential competitors revealed that the CsupGOBP2 protein has significant affinity to cis-11-hexadecenal (Z11-16:Ald), the main component of Ch. suppressalis pheromone and to laurinaldehyd and benzaldehyde, two general plant volatile aldehydes.

Molecular epidemiologic analysis of group A rotaviruses in adults and children with diarrhea in Wuhan city, China, 2000-2006.

To compare epidemiologic features and genetic characteristics of group A rotaviruses causing diarrhea in children and adults, a survey was conducted in Wuhan, China, during the period of Dec. 2000-May 2006. A total of 3839 stool specimens from diarrheal patients from eight hospitals were analyzed. Winter seasonality was observed for rotavirus diarrhea in both adults and children, showing overall rotavirus-positive rates of 9.0 and 23.9%, respectively. Throughout the study period, G3 was the most frequent G serotype in both adults and children (detection rates 86.2 and 87.8%, respectively), and was mostly associated with VP4 genotype P[8], VP 6 genotype II (subgroup II), and NSP4 genotype B. G3 rotaviruses were differentiated into eight electropherotypes, among which seven types were found in specimens from both adults and children. VP7 gene sequences of G3 rotaviruses from adults and children (6 and 4 strains, respectively), detected in different years and different hospitals, showed extremely high sequence identities (99-100%) to each other and to a few G3 rotavirus strains reported in Asia. However, lower sequence identities (82-96%) were observed to most of the human and animal G3 rotaviruses reported so far, including some Chinese strains. These findings indicate that in Wuhan, China, epidemic and genetic features of rotaviruses are similar in adults and children, and it has been suggested that G3 rotaviruses that might have originated from the same rotavirus were circulating among children and adults as prevailing viruses. In this study, two rotavirus strains, G9P[8] strain L169, derived from an adult, and G4P[6] strain R479, derived from a child, were isolated and genetically analyzed. The VP7 gene of L169 belongs to a major lineage of G9 rotaviruses that are globally widespread, but is distinct from G9 rotaviruses reported previously in China. The strain R479 had a VP7 gene which was divergent from most G4 human rotaviruses and showed an unusual dual subgroup specificity, I + II. The R479 VP6 gene does not belong to the main clusters of subgroup I and II rotaviruses phylogenetically, but is related to those of the porcine rotaviruses and some unusual human rotaviruses represented by the RMC321 strain isolated in eastern India.

Glossina austeni (Diptera: Glossinidae) eradicated on the island of Unguja, Zanzibar, using the sterile insect technique.

An area-wide integrated tsetse eradication project was initiated in Zanzibar in 1994 by the International Atomic Energy Agency and the governments of Tanzania and Zanzibar, to eradicate Glossina austeni Newstead from Unguja Island (Zanzibar) using the sterile insect technique. Suppression of the tsetse population on Unguja was initiated in 1988 by applying residual pyrethroids as a pour-on formulation to livestock and by the deployment of insecticide impregnated screens in some of the forested areas. This was followed by sequential releases of gamma-sterilized male flies by light aircraft. The flies, packaged in carton release containers, were dispersed twice a week along specific flight lines separated by a distance of 1-2 km. More than 8.5 million sterile male flies were released by air from August 1994 to December 1997. A sterile to indigenous male ratio of >50:1 was obtained in mid-1995 and it increased to >100:1 by the end of 1995. As a consequence the proportion of sampled young females (1-2 ovulations), with an egg in utero in embryonic arrest or an uterus empty as a result of expulsion of a dead embryo, increased from <25% in the 1st quarter to >70% in the last quarter of 1995. In addition, the age structure of the female population became significantly distorted in favor of old flies (> or = 4 ovulations) by the end of 1995. The apparent density of the indigenous fly population declined rapidly in the last quarter of 1995, followed by a population crash in the beginning of 1996. The last trapped indigenous male and female flies were found in weeks 32 and 36, 1996, respectively. Time for 6 fly generations elapsed between the last catch of an indigenous fly and the end of the sterile male releases in December 1997.

[Determination of alkyl alcohol polyoxyethylene ether by high temperature gas chromatography].

High temperature GC method has been developed for the separation and determination of alkyl alcohol polyoxyethylene ether (AEO). These AEO samples were separated on high temperature Al-coated fused-silica capillary column (0.1 micron bonded methyl silicon stationary phase, 25 m x 0.25 mm i.d.). The components of AEO sample were identified by GC/MS. The free alkyl alcohol and ethoxymer distribution of polyoxyethylene of AEO sample were determined by normalization method. The FID responses of typical components of AEO sample were determined, and their relative deviations were less than 4.1%. The recoveries of the free alkyl alcohol ranged from 96.5% to 98.1%. The relative standard deviations were less than 1.9%. In comparing with previous methods, this method is simple, fast and more reproducible.

Field responses of Glossina austeni to sticky panels on Unguja Island, Zanzibar.

Two designs of cross-shaped sticky panels (XT and XLP) were compared with the royal blue-white legpanel (LPBuWh) in the Jozani forest on Unguja Island as trapping devices for male Glossina austeni. Single coloured royal blue (XTBu) and bi-coloured royal blue-white XT (XTBuWh) caught more than twice as many male G. austeni as the LPBuWh, whereas single coloured black XT trapped significantly fewer flies (10%) than the control LPBuWh. XT's in various horizontal and diagonal blue-white configurations likewise trapped more flies than the LPBuWh, except a horizontally striped blue-white XT which trapped fivefold fewer flies than the LPBuWh. Cross-shaped LP in the blue-white (XLPBuWh) and black-white (XLPB1Wh) combination scored significantly better than the control LPBuWh. Similar fly numbers were trapped with XTBuWh and XLPBuWh. Long-term trapping data indicated that the XTBu, XTBuWh and XLPBuWh were three- to fourfold more effective in trapping female flies than the LPBuWh. The landing bias on bi-coloured panels was low in the blue-white but more pronounced in the blue-black and white-black combinations and was affected by the type of sticky panel used. A high proportion (49%) of the flies alighted on the bottom corners of the XTBu panel, but landing positions were more scattered if white was added. Increasing the width of the XTBu from 70 to 120 cm improved the catch by a factor of two as compared with standard sized XTBu. The effect of doubling the height of the XT on total fly catch was negligible. At present, it is the XTBu which can be recommended as the best trapping device for male and female G. austeni.

Vitamin A concentration in breast adipose tissue of breast cancer patients.

Vitamin A concentration in breast adipose tissue as a risk factor for breast cancer was studied by assessing the vitamin A concentration in breast adipose tissue of women with benign breast disease (n = 45) and of those with breast cancer (n = 36). No significant differences in vitamin A concentration between the two groups were found either in premenopausal or postmenopausal subjects. The same was true with the dietary intake of vitamin A in these two groups of women. The vitamin A concentration in breast adipose tissue had a significant correlation with the dietary intake in breast cancer cases only (r = 0.42, p < 0.01). The present results support the previous suggestion that vitamin A is unlikely to be related to the risk of breast cancer.

Microcomputer system for automatic identification of the Cryptococcus neoformans and its clinical application.

In this study, microcomputer image processing and pattern recognition technology, and the knowledge of morphology and optical characteristics of Cryptococcus neoformans were used for identification of Cryptococcus neoformans. Four groups of mice were lethally infected with standard strain, Wuhan strain, American B-2643 strain and Var. Shanghainesis of the Cryptococcus neoformans. The samples collected included mice brain, lung, kidney, liver, small intestine tissue and were observed under a light microscope. More than 600 images of the fungus were input into a microcomputer. A system of computer for automatic identification of the Cryptococcus neoformans was developed. The technique involved image preprocessing, image segmenting, coding of line-length on the edge, curve fitting, extracting of image feature, building of image library and feature data bank etc.. And then, 768 images of the clinical samples and other fungus samples whose morphological features tend to be confused with Cryptococcus neoformans were input into microcomputer and subjected to automatic identification. The Cryptococcus neoformans was accurately identified within 15 min, and the consistency rate with results of routine culture was 98%.

Fatty acid composition of breast adipose tissue in breast cancer patients and in patients with benign breast disease.

Fatty acid composition of triglycerides (TGs) and phospholipids (PLs) in breast adipose tissue was analyzed in 73 female breast cancer patients and 55 patients with benign breast disease. No differences were observed in the dietary intake of the major fatty acids (i.e., palmitic, stearic, oleic, and linoleic acids) or in the proportion of TGs and PLs in breast adipose tissue between the two groups. In postmenopausal women, however, the dietary intake of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) was significantly lower in the breast cancer patients than in patients with benign breast disease. Accordingly, the percentage of docosahexaenoic acid of PLs in breast adipose tissue was significantly lower in breast cancer patients than in patients with benign breast disease among postmenopausal women. The stage of the breast cancer did not contribute to the observed alterations of fatty acid composition of PLs. Consonant with the previous epidemiologic data, the present results suggest that intake of the long-chain n-3 fatty acids (mainly derived from fish) may have a protective effect against breast cancer, particularly in postmenopausal women.

[Pathogenesis of posterior capsular opacification after extracapsular cataract extraction].

Rabbit eyes were enucleated and examined by light and electronic microscope immediately after extracapsular surgery, and one day, one week, two weeks, one month and 6 months postoperatively. In addition, the technique of autoradiography was used to examine the status of cell hyperplasia of the lens epithelium. The results showed that posterior capsular opacification was due to migration of the lens epithelial cells onto the posterior capsule and transformation into fibroblasts. Fibrin exudate from postoperative uveitis was also a contributing factor.

[Follow-up for posterior capsular opacification after extracapsular cataract extraction in 263 eyes].

263 eyes (252 patients) were followed up after extracapsular cataract extraction for posterior capsular opacifications. It was found that the incidence of this complication was higher after longer periods of follow-up, and also more frequent in younger patients. The etiology is due to migration and fibrosis of hyperplastic epithelial cells. Postoperative uveitis may be a contribution factor. Nd: YAG laser is the best means to perforate the posterior capsular opacification.

Effects of sodium iodate on experimental subretinal neovascularization in the primate.

We investigated the role of the retinal pigment epithelium (RPE) in the development of subretinal neovascularization (SRN) induced by bilateral laser photocoagulation of the retinas in monkeys. RPE function was affected by a unilateral carotid injection of sodium iodate, a selective RPE toxin, before laser photocoagulation. This caused severe RPE damage in the ipsilateral eye, but did not affect the contralateral eye. After laser photocoagulation no SRN was demonstrable clinically or histopathologically in the ipsilateral eye; in contrast, active SRN showing extensive fluorescein leakage was found in the contralateral eye. These results suggest that RPE may play a role in the development of laser-induced SRN.

Image processing by microcomputer on ultrastructure of griseofulvin-resistant fungi in favus.

The ultrastructure of 5 griseofulvin-resistant fungi of favus was studied by image processing with microcomputer. It was found that the cell walls of the fungi consisted of 8 layers, and the inner layer containing cytoplasm was loose. It was also found that all structures within the cytoplasm possessed a 1-3 layers integral envelope with chromatins in the nucleus. These might be contributing factors in the development of resistance to griseofulvin. This multiple-layered, thick cell wall might act as a barrier responsible for the impermeability of the cell to griseofulvin.

Anti-shock effects of synthetic effective compositions of fructus aurantii immaturus. Experimental study and clinical observation.

Satisfactory results have been obtained in treating infective shock with injection of natural Fructus Aurantii immaturus (nat-FAI). The results of animal experiments and clinical observations on the anti-shock effects of the synthetic effective compositions of Fructus Aurantii immaturus (syn-FAI) are reported. The cardiac output increased from 0.53 to 0.87 L/min (P less than 0.01), and the cardiac index increased from 0.99 to 1.63 L/m2/min (P less than 0.01) in the endotoxic shock dogs after the treatment with syn-FAI. At the same time the blood stream in bulbar conjunctiva became accelerated and the dilated microvessels began to get smaller in most dogs. Of fifty children with infective shock treated with syn-FAI, forty-eight showed curative effects, with a total effective rate of 96%. The anti-shock effective compositions in FAI have been proved to be synephrine and N-methyltyrosamine. Moreover, syn-FAI has shown a more stable property, less side-effects and better clinical results than nat-FAI.

Experimental subretinal neovascularization in the rabbit.

Subretinal neovascularization (SRN) in the rabbit was induced by subretinal injection of vitreous without rupture of Bruch's membrane. Eight of 26 eyes developed SRN. The incidence of SRN rose from 33% to 57% in a period of 4-40 weeks. Because of the absence of any fluorescein angiographic indication of SRN, these occult new vessels were identified by light and transmission electron microscopy. Histological examination showed that these newly formed vessels are composed of continuous capillaries with the morphologic characteristics of choriocapillaris, including diaphragmed fenestrations, basement membranes, and junctional complexes. The new vessels originated from the choriocapillaris and penetrated through Bruch's membrane into the subretinal space, where they were associated with the degenerated sensory retina and proliferating glial and/or RPE cells. This experiment provides a model of SRN without breaks in Bruch's membrane.