Use of nimotuzumab combined with cisplatin in treatment of nasopharyngeal carcinoma and its effect on expressions of VEGF and MMP-2

Purpose This paper aims to observe the expressions of VEGF and MMP-2 in patients with nasopharyngeal carcinoma treated by nimotuzumab combined with cisplatin. Methods Altogether, 104 patients with nasopharyngeal carcinoma treated in our hospital from April 2014 to August 2016 were selected as research subjects. Among them, 50 patients treated with cisplatin were divided into a control group and 54 patients treated with nimotuzumab combined with cisplatin were divided into an observation group. The two groups of patients were compared in terms of efficacy after treatment and incidence of adverse reactions. Changes of serum VEGF and MMP-2 concentrations before and after treatment were detected using enzyme-linked immunosorbent assay (ELISA), and the 3-year overall survival (OS) of patients was observed. Results Compared with the control group, patients in the observation group had significantly higher total remission rate (RR) (P < 0.05) and significantly lower incidence of adverse reactions (P < 0.05). Before treatment, there was no significant difference between the observation and control groups in the concentrations of VEGF and MMP-2 (P > 0.05). After treatment, the concentrations in the two groups were significantly lower than those before treatment (P < 0.05), and the concentrations in the observation group were significantly lower than those in the control group (P < 0.05). There was no significant difference in the 3-year OS between the observation and control groups (P > 0.05). Conclusions Nimotuzumab combined with cisplatin could improve the conditions of patients with nasopharyngeal carcinoma. After treatment, the expression of VEGF and MMP-2 decreased significantly. We speculated that it improves the survival rate of patients by reducing the expression of VEGF and MMP-2 (AU)

Use of nimotuzumab combined with cisplatin in treatment of nasopharyngeal carcinoma and its effect on expressions of VEGF and MMP-2.

PURPOSE: This paper aims to observe the expressions of VEGF and MMP-2 in patients with nasopharyngeal carcinoma treated by nimotuzumab combined with cisplatin. METHODS: Altogether, 104 patients with nasopharyngeal carcinoma treated in our hospital from April 2014 to August 2016 were selected as research subjects. Among them, 50 patients treated with cisplatin were divided into a control group and 54 patients treated with nimotuzumab combined with cisplatin were divided into an observation group. The two groups of patients were compared in terms of efficacy after treatment and incidence of adverse reactions. Changes of serum VEGF and MMP-2 concentrations before and after treatment were detected using enzyme-linked immunosorbent assay (ELISA), and the 3-year overall survival (OS) of patients was observed. RESULTS: Compared with the control group, patients in the observation group had significantly higher total remission rate (RR) (P < 0.05) and significantly lower incidence of adverse reactions (P < 0.05). Before treatment, there was no significant difference between the observation and control groups in the concentrations of VEGF and MMP-2 (P > 0.05). After treatment, the concentrations in the two groups were significantly lower than those before treatment (P < 0.05), and the concentrations in the observation group were significantly lower than those in the control group (P < 0.05). There was no significant difference in the 3-year OS between the observation and control groups (P > 0.05). CONCLUSIONS: Nimotuzumab combined with cisplatin could improve the conditions of patients with nasopharyngeal carcinoma. After treatment, the expression of VEGF and MMP-2 decreased significantly. We speculated that it improves the survival rate of patients by reducing the expression of VEGF and MMP-2.

[Analysis of the efficacy of the Ponseti method for treatment of secondary clubfoot in young children with tethered cord syndrome].

Objective: To explore the feasibility of Ponseti method in treatment of secondary clubfoot in young children with Tethered Cord Syndrome(TCS). Methods: The clinical data of 53 young children with clubfeet treated with Ponseti method from March 2014 to March 2017 at Department of Pediatric Orthopedics, the Third Affiliated Hospital of Zhengzhou University were analyzed retrospectively. These patients were divided into TCS group and Idiopathic group according to the etiology. There were 19 patients (33 feet) in TCS group,with an mean age of 2.8 months(range:0.2 to 24.0 months), including 13 males and 6 females, 5 patients with unilateral clubfeet and 14 patients with bilateral clubfeet. There were 34 patients (45 feet) in idiopathic group, with an mean age of 3.1 months(range: 0.1 to 21.0 months), including 18 males and 16 females, 23 patients with unilateral clubfeet and 11 patients with bilateral clubfeet. All the children received casts correction according to Ponseti method, and were followed up at 3 weeks, 3 months, 6 months and every 6 months after the Achilles tendon tenotomy or the last cast correction. Complications were recorded and therapeutic effect was evaluated of these children by Dimeglio Scoring System and the International Clubfoot Study Group (ICFSG) at the last follow-up. Independent t test, Mann-Witney U test or χ(2) test were used to compare the indicators of the two groups. Results: The number of plaster fixation in TCS group was (6.1±2.0) times, and that of idiopathic group was (4.8±1.0) times(t=3.482, P<0.01).In TCS group, 22 feet treated with Achilles tendon transection and that of idiopathic group was 40 feet(χ(2)=0.279, P=0.598). There were 18 cases recurrence in TCS group and 8 cases in Idiopathic group (t=11.149, P<0.01). In TCS group, 16 cases (27 feet) completed the initial correction, the success rate was 60.6% (27/33), 3 cases (6 feet) could not correct the deformity after 9 to 10 times of plaster fixation, and then underwent soft tissue release.In idiopathic group, 34 cases (45 feet) achieved initial correction after Ponseti treatment(χ(2)=6.488, P=0.011).At the last follow up, there were 5 cases (9 feet) in TCS group and 2 cases (2 feet) in idiopathic group underwent soft tissue release(χ(2)=6.110, P=0.013). The classification grade of ICFSG score of the two groups without soft tissue release were (2.1±0.6) and (1.8±0.7), the difference was not statistically significant (t=1.765, P=0.082). All the children had no skin ulceration, bedsores, skin allergy and other complications. Conclusion: Ponseti method is effective in the treatment of clubfoot secondary to TCS, and the functional recovery is similar to that of children with idiopathic clubfoot.

Microvesicles containing microRNA-216a secreted by tubular epithelial cells participate in renal interstitial fibrosis through activating PTEN/AKT pathway.

OBJECTIVE: The aim of this study was to elucidate the role of microRNA-216a in microvesicles (MVs) during the process of renal interstitial fibrosis and to investigate its underlying mechanism. MATERIALS AND METHODS: Unilateral ureteral occlusion (UUO) model was first established in mice, and kidney tissues and urine in the obscured kidney were collected. NRK-52E cells were induced with 5 ng/mL transforming growth factor-ß1 (TGF-ß1) for constructing the renal interstitial fibrosis model in vitro. Subsequently, the expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (FN) in NRK-52E cells induced with or without TGF-ß1 were determined, respectively. The culture medium was collected from NRK-52E cells of the control group (without TGF-ß1 induction) and the TGF-ß1 group (TGF-ß1 induction), and MVs were observed. Afterward, NRK-52E cells were treated with MVs isolated from the control group or the TGF-ß1 group, followed by detecting the expressions of E-cadherin, α-SMA and FN. Meanwhile, the expression levels of CD63, microRNA-216a, PTEN and p-AKT were determined as well. The microRNA-216 level in kidney tissues and urine of UUO mice were determined. Furthermore, the expressions of PTEN and p-AKT in mouse kidney tissues were accessed by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RESULTS: TGF-ß1 induction in NRK-52E cells gradually downregulated E-cadherin, whereas upregulated α-SMA and FN with the prolongation of induction time. MVs isolated from the culture medium of the TGF-ß1 group downregulated E-cadherin, and upregulated FN and α-SMA. The expression levels of CD63 and microRNA-216a were markedly higher in the TGF-ß1 group compared with the control group. Downregulated PTEN and upregulated p-AKT were observed in TGF-ß1-induced cells at both mRNA and protein levels. Besides, microRNA-216a expression in mouse kidney tissues and urine from obscured kidney was remarkably increased with the prolongation of UUO. Consistent with those in NRK-52E cells, the protein level of PTEN was significantly decreased, whereas p-AKT was markedly increased with the prolongation of UUO. CONCLUSIONS: MVs containing microRNA-216a secreted by injured proximal tubular epithelial cells participate in renal interstitial fibrosis by activating the PTEN/AKT pathway.

¹H-MRS before and after resuscitation following selective cerebral ultra-profound hypothermic blood flow occlusion in monkeys.

We investigated the effect of selective cerebral ultra-profound hypothermic blood flow occlusion on brain tissue and cell metabolism to ascertain the efficacy and safety of selective deep hypothermic technologies using proton magnetic resonance spectroscopy ((1)H-MRS). The bilateral carotid artery was blocked at room temperature for 10 min. Other neck vessels were then blocked through cold perfusion of the internal carotid artery and reflux of the ipsilateral jugular vein. Thus, selective cerebral extracorporeal circulation was established. Brain temperature was reduced to 15.1° ± 0.9°C. After 60 min, cerebral blood flow recovered naturally. Routine magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI), and (1)H-MRS examination of the bilateral frontal cortex and basal ganglia were performed prior to surgery and 4, 24, 72 h, 21 days after recovery. The formants and areas under the curve (AUC) of N-acetyl aspartate (NAA), choline (Cho), creatine/phosphocreatine (Cr/Cr2) were analyzed using 1H-MRS. The pre- and postoperative AUC of NAA and Cho at different time points were compared. Conventional MRI and DWI showed no abnormal signal changes in the brain parenchyma or right basal ganglia before and after surgery (P > 0.05). There was no significant difference in the ratio between NAA/(Cr+Cr2) and Cho/(Cr+Cr2) before and after surgery in the bilateral basal ganglia and frontoparietal regions of the cortex (P > 0.05). Quantitative (1)H-MRS showed that selective deep cerebral hypothermia significantly improved the brain's tolerance to ischemia and hypoxia. Our results could provide a better understanding of the efficacy and safety of selective deep hypothermia and blood flow occlusion.

Changes in hippocampal ultrastructure and vimentin expression in rhesus monkeys following selective deep hypothermia and blood occlusion.

Previous studies have shown that selective cerebral profound hypothermia combined with antegrade cerebral perfusion can improve resistance to cerebral hypoxia-ischemia in monkeys. The aim of this study was to observe the effect of selective cerebral profound hypothermia on the ultrastructure and vimentin expression in monkey hippocampi after severe cerebral ischemia. Eight healthy adult rhesus monkeys were randomly divided into two groups: profound hypothermia (N = 5) and normothermia (N = 3). Monkeys in the profound hypothermia group underwent bilateral carotid artery and jugular vein occlusion for 10 minutes at room temperature. Ringer's solution at 4°C was then perfused through the right internal carotid artery and out of the right jugular vein, maintaining the brain temperature below 18°C. Sixty minutes later, cerebral blood flow was restored. The normothermia group underwent all procedures with the exception that the Ringer's solution was 37°C during perfusion. All animals in the profound hypothermia group were successfully resuscitated. No significant abnormalities of hippocampal morphology or ultrastructure were observed. In contrast, no monkeys were alive after perfusion in the normothermia group and they had abnormal hippocampal morphology and ultrastructure to different extents. Vimentin expression in the hippocampus was significantly lower in the profound hypothermia group (47.88% ± 1.66) than the normothermia group (79.51% ± 1.00; P < 0.01). We conclude that selective cerebral profound hypothermia following 10-min occlusion of the bilateral common carotid arteries was able to downregulate vimentin expression in the hippocampus and protect it from severe cerebral ischemia.

Insulin-like growth factor binding protein-3 (IGFBP-3) genetic variant and the risk of esophageal squamous cell carcinoma in a Chinese population.

Insulin-like growth factor binding protein-3 (IGFBP-3) exerts anti-proliferative or pro-apoptotic effects through IGF-dependent as well as IGF-independent mechanisms in vitro. The purpose of this study was to examine the association between genetic variants in IGFBP-3 (rs2270628) and the risk of esophageal squamous cell carcinoma (ESCC) in a Chinese Han population. Five hundred ESCC cases and 500 cancer-free controls of the Chinese Han population were involved in this study. The IGFBP-3 single-nucleotide polymorphism (SNP) rs2270628 was genotyped and the estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for its association with the risk of ESCC were determined using unconditional logistic regression analysis. Compared with the rs2270628 CC genotype, TT genotype was associated with a significantly increased ESCC risk with OR (95%CI) of 2.07 (1.05-4.09), but CT genotype was not (OR = 1.25, 95%CI =0.94-1.66). IGFBP-3 SNP rs2270628 may contribute to the risk of ESCC in the Chinese Han population.

First Report of Pindo Palm Heart Rot Caused by Ceratocystis paradoxa in China.

On January 12th, 2012, a novel disease with an incidence of 50% was discovered in Pindo palm Butia capitata (Mart.) Becc from the Coconut Grant View Garden (19°33.137' N, 110°47.482' E) located in Wenchang, Hainan Province. Diseased leaflets at the base of the rotted heart leaves had reddish brown lesions; when the infection progressed, the leaves turned yellow and became blighted from the inner to the outer part of the crown. Once the growing point was destroyed, the entire tree ultimately died. Tissues from the edges of lesions from diseased leaflet samples were placed onto potato dextrose agar (PDA) and incubated at 25°C for 3 days. The color of colonies of five isolates obtained turned from white to black in 48 h. The optimum temperature for mycelium growth was from 20 to 30°C, and no growth occurred at temperatures higher than 40°C or lower than 5°C (n = 5). The cylindrical colorless to pale brown conidia were 7.5 to 17.5 µm long × 5.0 to 7.5 µm wide (n = 100); oval black chlamydospores were 12.5 to 22.5 × 7.5 to 15.0 µm (n = 100). The sequence (497 bp) of the internal transcribed spacer (ITS) region of the representative isolate BX3 (China Center for Type Culture Collection No. CCTCC AF2014002) was amplified using primer pair ITS1/ITS4 (GenBank Accession No. KF939052) and shared 99% sequence identity with Ceratocystis paradoxa strain xie331-4 (JQ039332). Based upon these biological characteristics and ITS sequence, this pathogen was identified as C. paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn.) (3). Pathogenicity tests were conducted on 8-cm-long sections of young leaflets excised from a 12-year-old pindo palm tree. One side of the midrib of 10 sections was wounded with a sterilized scalpel at the center and the other side was non-wounded, then a PDA plug (4 to 6 × 4 to 6 mm) from the edge of an actively growing colony of BX3 incubated for 3 days were inoculated onto each wounded or non-wounded site. As controls, plain PDA plugs were placed on wounded and non-wounded spots of another 10 sections following the above procedure. Pathogenicity was tested twice. Each inoculated section was then put into a 9-cm petri dish in which two filter papers (Φ = 9 cm) were placed and 8 ml of sterile water were added to maintain high humidity, and then all dishes were placed in a dark incubator at 25°C. After 5 days, typical symptoms developed only on the wounded points inoculated with mycelium plugs. C. paradoxa was re-isolated from the margins of the expanding lesions. C. paradoxa causing fruit rot of B. capitata was reported in Uruguay (2), but to our knowledge, there are no previous reports of this species in China or infecting leaves of B. capitata worldwide (1). We report here a new Ceratocystis disease on B. capitata, and it was named as pindo palm heart rot based on its symptoms. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , Feb 21, 2014. (2) V. Gepp et al. New Dis. Rep. 27:12, 2013. (3) F. Y. Yu et al. Plant Dis. 96:290, 2012.

First Report of Leaf Spot Disease in Coconut Seedling Caused by Bipolaris setariae in China.

Coconut (Cocos nucifera L.), an important oilseed as well as a multipurpose perennial plantation crop, is distributed and planted in humid tropical areas. In October 2012, a new leaf spot disease was observed on 3-year-old coconut seedlings in Wenchang, Hainan Province, China. The symptom first appeared as spindly or elliptical and brown flecks with water-soaked lesions that became yellow with the progress of the disease. In the later stage of the disease, the lesions merged together, gradually expanding to the leaf apex. In recent years, the disease has been prevalent in all the nursery gardens surveyed. Once young leaves got infected and nearly all the leaves of the tree showed diseased symptoms, the coconut eventually became defoliated. The pathogen was isolated from the lesion margin, surface sterilized with 75% ethanol and 0.1% mercury bichloride, washed by sterile distilled water, and then placed excising pieces of leaves from the leision margin onto potato dextrose agar (PDA). Plates were incubated at 25°C for 4 days. After 7 days, the colony was grayish black and produced black pigment in the medium. Aerial mycelium was fluffy, septate, and branched, the conidiophores were slightly flexuous or straight, 5 to 11 µm thick, and produced curved, spindle-shaped, or fusiform, septate conidia with 4 to 10 septa, measuring 39 to 86 × 9 to 16 µm, with a slightly protuberant hilum, truncated. Based on the symptoms and mycelial and conidial characters above, the fungus was identified as Bipolaris setariae (1). The pathogenicity was established and repeated for six times by following Koch's postulates. Two 1-year-old coconut seedlings were washed with sterilized water and six leaves were wounded with a sterile needle and then inoculated by spraying them with a suspension of conidia of the isolate. The seedlings were kept in two incubators at 25°C for 12 days. Inoculated leaves showed typical symptoms similar to those described above. The pathogen was re-isolated from inoculated leaves. Morphological characteristics were identical to the original isolated fungus. In contrast, the control leaves did not show any symptoms. The genomic DNA of this fungus was extracted, amplification of the internal transcribed spacer (ITS) region was performed with primer ITS1 and ITS4, and the purified PCR product was sequenced (GenBank Accession No. KJ605157). BLASTn analysis revealed 99% sequence similarity with four B. setariae isolates (HE792936.1, JX462256, GU073108.1, and FJ606786.1). Morphologic characters and sequence analysis of the ITS rDNA confirmed that the pathogen was B. setariae. Bipolaris incurvata has been reported causing disease on coconut (2), but B. setariae was not previously reported on coconut. So far, this is the first report of B. setariae caused coconut seedling leaf spot disease in Hainan, China. References: (1) K. C. da Cunha et al. J. Clin. Microbiol. 50:4061, 2012. (2) A. Kamalakannan et al. New Dis. Rep. 12:18, 2005.

First Report of Leaf Spot of Tea Oil Camellia (Camellia oleifera) Caused by Lasiodiplodia theobromae in China.

Tea oil camellia (Camellia oleifera Abel.), one of the most famous woody oil plants, is distributed and cultivated widely in central and southern China for its strong adaptability. In September 2013, tea oil camellia plants with severe leaf spots were observed in commercial production fields located in Wenchang, Hainan Province. Spots were initially chlorotic, became necrotic and black with a chlorotic halo, developing to cover the entire width of the leaves, and leading to leaf death. Isolations were performed by excising pieces of symptomatic leaves from the lesion margin, surface sterilized with 90% ethanol and 0.6% sodium hypochlorite, and then placed them on potato dextrose agar (PDA). Plates were incubated in a sterile chamber at 26 ± 2°C for 2 days. A fungus was consistently isolated on PDA from all 23 diseased leaf samples. Pure cultures were obtained by monosporic culture technique. After 2 to 3 days of incubation at 26 ± 2°C with a 12-h photoperiod, the fungus initially produced white colonies with dense aerial mycelia, which later turned black (6 to 7 days). The mycelium was fast spreading, branched, and septate. Pycnidia were black, globose, ostiolate, and produced in stroma on the medium surface after 28 days at the same culture conditions as above. Conidia were initially unicellular, subovoid, hyaline, thick-walled with granular content, and 19.8 to 28.9 × 11.5 to 15.7 µm (avg. 25.1 × 13.5 µm). Mature conidia were one-septate and dark brown with longitudinal striations. These observed morphological features suggested that the fungus possessed the same characteristics as previously described for Lasiodiplodia theobromae (Pat.) Griffon & Maubl (syn = Botryodiplodia theobromae) (2). For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences submitted to GenBank were KF811055 for ITS region; KJ639047 for ß-tubulin; and KJ639048 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank (NR_111174, EU673110, and AY640258). Hence, both morphological and molecular characteristics confirmed the fungus as L. theobromae. To confirm fungal pathogenicity, ten 1-year-old healthy plants of C. oleifera were inoculated with the fungus. Mycelial plugs (5 mm) taken from a 7-day-old colony growing on PDA were deposited on wounds with a sterilized knife on leaves and covered with moist cotton. Ten additional control plants were treated similarly but with sterile PDA plugs. Plants were maintained in a moist chamber at 26 ± 2°C for 3 days and then in a greenhouse at 25°C and 40% relative humidity. All the inoculated plants produced typical leaf spot symptoms 3 weeks after inoculation. The fungus was consistently re-isolated from all inoculated plants. Control plants did not show any symptoms. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (1,2), but not previously reported causing disease on C. oleifera. To our knowledge, this is the first report worldwide of leaf spot of C. oleifera caused by L. theobromae. References: (1) S. Mohali et al. For. Pathol. 35:385, 2005. (2) E. Punithalingam. Page 519 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, UK, 1976.

First Report of Burkholderia andropogonis Causing Bacterial Leaf Spot of Betel Palm in Hainan Province, China.

In May 2009, a severe bacterial disease of arecanut (Areca catechu L.) with an incidence of 100% was observed in a plantation of about 8,400 plants in Wenchang City, Hainan Province, China (19°47.171' N, 110°54.335' E). Symptoms consisted of small circular to elongated brown lesions, ranging from 1 to 105 mm in length and 1 to 21 mm in width, surrounded by yellow halos. White colonies, without fluorescent or diffusible pigments, were consistently recovered on King's B Medium plates from lesions surface-sterilized in 70% ethyl alcohol for 1 min. All isolates were gram-negative and each had a single, polar, sheathed flagellum. Isolates were identified as a Burkholderia sp. based on physiological and biochemical tests: oxidase and catalase positive, negative for arginine dihydrolase, gelatin hydrolysis and starch hydrolysis, and negative for acid production from levan (1,3). Sequences (approx. 1,400 bp each) of the 16S rRNA gene amplified from four isolates using primer pair 27F/1492R (2) (GenBank Accession Nos. JX415481, JX415479, JX415482, and JX415483) shared 99% sequence identity with that of Burkholderia andropogonis strain 6369 (DQ786951). Representative isolates Y11 (China General Microbiological Culture Collection Center No. CGMCC 1.12337), Y30 (CGMCC 1.12338), W15, and W20 were compared with B. andropogonis strain NCPPB No. 1012 and all caused a hypersensitive reaction on leaves of Nicotiana benthamiana. Isolate pathogenicity was tested twice with a total of three replications per isolate. Two young leaves each of 2-year-old arecanut plants were infiltrated with a bacterial suspension of 108 CFU/ml, then covered individually with plastic bags for 48 h, and incubated at 100% relative humidity with 16 h of daylight at 25°C by day and 8 h of darkness at 20°C by night. After 7 days, small water-soaked spots with yellow halos were observed and 60 days after inoculation, lesions developed similar to those caused by B. andropogonis in the field. Koch's postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants. These bacteria were identical to inoculated strains in colony morphology and sequences of the 16S ribosomal RNA gene. To our knowledge, this is the first report of B. andropogonis infection on betel in Hainan Province, mainland China. This disease was first reported in Taiwan, a province of China. Conditions of high humidity and high temperature support disease outbreaks and infection can result in severe economic losses. In 2012, this disease also appeared on a number of plantations located in other counties. As betel is, economically, the second most important crop in Hainan Province, measures should be required to control this disease, especially in typhoon seasons. References: (1) S. H. Hseu et al. Plant Pathol. Bull. 16:131, 2007. (2) D. J. Lane. In: E. Stackebrandt, et al. Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom, pp. 115-175, 1991. (3) X. Li and S. H. De Boer. Plant Dis. 89:1132. 2005.

First Report of Stem Bleeding in Coconut Caused by Ceratocystis paradoxa in Hainan, China.

Stem bleeding of coconut was discovered in 2009 in Hainan, China. Affected trunk areas exhibited dark discoloration and a reddish brown or rust-colored liquid bleeding from different points. Stem tissues under the lesions rotted and became brownish yellow to black. Affected plants died within 3 to 4 months after stem symptoms first appeared. Stem bleeding of coconut is known to occur in production areas worldwide. The disease was first reported in Sri Lanka (1), caused severe damage to PB-121 hybrids in Indonesia (2), and is now known to occur in many other coconut-producing countries. However, to our knowledge, this is the first report of the disease in China. A fungus was isolated from lesion margins of diseased coconut trees. Colonies on potato dextrose agar (PDA) were white, became black 1 to 2 days later, and emitted a strong, fruity aroma. The fungus produced conidia, which were cylindrical, colorless to pale brown, and 6.9 to 14.9 × 3.1 to 6.0 µm, and oval, black chlamydospores that were 7.9 to 19.4 × 4.6 to 11.0 µm. The optimum temperature for mycelial growth ranged from 25 to 35°C and it did not grow at temperatures lower than 5°C or higher than 40°C. On the basis of these characteristics, the fungus was identified as Ceratocystis paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn). The internal transcribed spacer (ITS) region was amplified from genomic DNA with primers ITS1 and ITS4 and the PCR products were sequenced (GenBank Accession No. JQ039332). BLAST analysis showed 99% sequence similarity with C. paradoxa (GenBank Accession No. HQ248205.1). Pathogenicity of the fungus was tested by inoculating 10, 3-year-old coconut trees of the cv. green tall at the 12-leaf stage in the field. Agar plugs (5 mm in diameter) from the periphery of 7-day-old C. paradoxa colonies grown on PDA were placed on healthy trunks, rachis, and leaves, which were either wounded or unwounded. Wounds were made with a sterilized cork borer. Sites of the inoculations were wrapped with plastic tape to prevent desiccation; the experiment was repeated three times. Controls received plain PDA discs. Two weeks after inoculation, characteristic rusty brown lesions appeared only on wounded plants that were inoculated with the fungus. A brownish liquid oozed from the points of inoculation. Controls did not show signs of disease development. C. paradoxa was reisolated from the diseased tissues. Infection occurred on wounded sites only, suggesting that wounds may be required for infection. To prevent stem bleeding of coconut trees by C. paradoxa, vigilant cultural practices must be maintained to avoid causing wounds on the trees. References: (1) S. A. Alfieri. Plant Pathol. Circular No. 53. Florida Department of Agriculture Division of Plant Industry, 1967. (2) D. R. N. Warwick and E. E. M. Passos. Trop. Plant Pathol. 34:175, 2009.