ABSTRACT
Rubus cochinchinensis, an important traditional Chinese medicine in China is used to treat rheumatic arthralgia, bruises and lumbocrural pain (He et al.2005). In January 2022, yellow leaves of R. cochinchinensis were found in Tunchang City, Hainan Province, a tropical island in China. Chlorosis spread along the direction of vascular tissue while the leaf veins remain green (Fig. 1). In addition, the leaves were slightly shrunken and the growth vigor is poor (Fig. 1). By survey, we found the incidence of this disease was about 30%. Three etiolated samples and three healthy samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Using nested PCR method, phytoplasma universal primers P1 / P7 (Schneider et al., 1995) and R16F2n / R16R2 (Lee et al. 1993) were used to amplified phytoplasma 16S rDNA gene. Primers rp F1 / R1 (Lee et al. 1998) and rp F2 / R2 (Martini et al. 2007) were used to amplified rp gene. 16S rDNA gene and rp gene fragments were amplified from three leaf etiolated samples, but not from healthy samples. The amplified fragments were cloned and sequenced, and the sequences were assembled by DNASTAR11. By sequence alignment, we found the obtained 16S rDNA and rp gene sequences of three leaf etiolated samples were same. The length of 16S rDNA fragment was 1237 bp (accession number: ON944105) and the length of rp gene fragment was 1212 bp (accession number: ON960069). The phytoplasma strain was named as 'R. cochinchinensis' yellows leaf phytoplasma (RcT), RcT-HN1 strain. The 16S rDNA gene sequence of RcT-HN1is 99.8% consistent with 16SrI-B subgroup members such as the 'Brassica napus' dwarf phytoplasma strain WH3 (MG599470.1), Chinaberry yellows phytoplasma strain LJM-1(KX683297.1) and Arecanut yellow leaf disease phytoplasma strain B165 (FJ694685.1). The rp gene sequence of RcT-HN1 is 100% consistent with rpI-B subgroup members such as the 'Salix tetradenia' witches'-broom phytoplasma strain YM-1 (KC117314.1) and Chinaberry witches'-broom phytoplasma strain Hainan (EU348781.1). The phylogenetic tree analysis, based on concatenated 16S rDNA-rp gene sequence of same group phytoplasma by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value, were performed (Kumar et al., 2016). The results showed that RcT-HN1 phytoplasma strain formed a subclade in aster yellows group B subgroup (Fig. 2). The virtual RFLP analysis based on the 16S rRNA gene fragment of RcT-HN1 phytoplasma strain was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009). The results showed that the phytoplasma strain was same as the reference pattern of the onion yellows phytoplasma of 16SrI-B (GenBank accession: AP006628), and the similarity coefficient was 1.00. This is the first report that 16SrI-B subgroup related phytoplasma infected R. cochinchinensis and caused yellows symptoms in China. The discovery of the disease is helpful to the study of the spread of phytoplasma-related diseases and protect R. cochinchinensis resources.
ABSTRACT
Carica papaya Linn, belonging to the Caricaceae family, is an economic and medicinal plant, which is widely cultivated in tropical and subtropical countries (Soib et al., 2020). Beginning in 2021, abnormal symptoms of Carica papaya exhibiting leaf yellow, crinkle and leaflet were found in Wanning city of Hainan Province, China. The diseased symptoms of the plant, with about 20 % incidence in the sampling regions, were suspected to be induced by phytoplasma, a phloem-limited and could not be cultured in vitro prokaryotic pathogen. Total DNAs were extracted from 0.10 g fresh leaves of symptomatic or asymptomatic Carica papaya using CTAB DNA extraction method (Doyle and Doyle, 1990). PCR reactions were performed using primers R16mF2/R16mR1 (Gundersen and Lee, 1996), secAfor1/secArev3 (Hodgetts et al., 2008) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA, secA and groEL gene fragments. PCR products of the 16S rRNA, secA and groEL gene target fragments of phytoplasma were obtained from the DNA of eight diseased Carica papaya samples whereas not from the DNA of the asymptomatic plant samples. The PCR amplicons of the three genes were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) and the sequences data were deposited in GenBank. The 16S rRNA, secAgroEL gene of phytoplasma was in length of 1326 (GenBank accession: OL625608), 716 (OL630087) and 1300 (OL630088) bp separately, putatively encoding 238 (secA) and 432 (groEL) amino acids sequence. The phytoplasma strain was named as Carica papaya yellow phytoplasma (CpY), CpY-hnwn strain. A blast search based on 16Sr RNA gene of CpY-hnwn showed 100 % sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628), Chinaberry witches'-broom phytoplasma (CWB) strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), Rapeseed phyllody phytoplasma isolate RP166 (CP055264). RFLP analysis based on the 16S rRNA gene fragment of CpY-hnwn was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009) indicated that the phytoplasma strain is a member of 16SrI-B subgroup. Blast search based on secA gene of CpY-hnwn showed 100 % sequence identity with that of CWB strains CWB-gdgz (KP662182), CWB-jxnc (KP662180) and CWB-fjya (KP662178) belonging to 16SrI-B subgroup. Blast search based on groEL gene of CpY-hnwn showed 99.77 % sequence identity with that of mulberry dwarf phytoplasma (AB124809) and 99.69 % sequence identity with that of Onion yellows phytoplasma strain OY-M (AP006628) and Rapeseed phyllody phytoplasma isolate RP166 (CP055264). Phylogenetic analysis based on the 16S rRNA gene fragments performed by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) indicated that the CpY-hnwn phytoplasma strain was clustered into one clade with the phytoplasma strains of OY-M (AP006628), RP166 (CP055264), CWB-hnsy1 (KP662119), CWB-hnsy2 (KP662120) and areca palm yellow leaf (KF728948), with 100 % bootstrap value. To our knowledge, this is the first report that a 16SrI-B subgroup phytoplasma infects Carica papaya in Hainan Province, a tropical island of China. Carica papaya was previously reported to be infected by 16SrXII-O subgroup phytoplasmas in Nigeria (Kazeem et al., 2021), 16SrII-U subgroup in Hainan Province of China (Yang et al., 2016). The findings in this study indicated that one plant couldthe phytoplasmas belonging to different 16Sr groups, which would be beneficial to the specific detection of the pathogens and the epidemic monitoring of the related diseases. References: Doyle, J.J. and Doyle, J.L. 1990. Focus 12: 13-15. Felsenstein, J. 1985. Evolution 39: 783-791. Gundersen, D.E. and Lee, I.M. 1996. Phytopath. Medit. 35: 144-151. Hodgetts, J., et al. 2008. Int. J. Syst. Evol. Microbiol. 58: 1826-1837. Kazeem, S.A., et al. 2021. Crop Prot. 148: 105731. Kumar, S., et al. 2016. Mol. Biol. Evol. 33: 1870-1874. Mitrovic, J., et al. 2011. Ann. Appl. Biol. 159: 41-48. Soib, H.H., et al. 2020. Molecules, 25: 517. Yang, Y., et al. 2016. Int. J. Syst. Evol. Microbiol. 66: 3485-3491. Zhao, Y., et al. 2009. Int. J. Syst. Evol. Microbiol. 59: 2582-2593.
ABSTRACT
Waltheria indica L. is a kind of medicinal plants belonging to the family of Sterculiaceae distributed in China, which extracts with many active compounds used for treatment of rheumatism and sore pains (Hua et al., 2019). During September to November 2020, the plants showing abnormal symptoms including floral virescence, leaf chlorosis and leaflet, as shown in Fig.1, were found in Dingan county of Hainan province, China, with about 70% incidence. The disease symptoms which were suspected to be infected by the phytoplasma, a phloem-limited cell-wall-less prokaryotic pathogen could not be cultured in vitro, severely impacted Waltheria indica growth resulting in financial loss and ecological damage in the location. For identification of the causal pathogen, the total DNA of symptom or symptomless Waltheria indica samples were extracted using 0.10 g fresh plant tissues using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 (Lee et al., 1993) and AYgroelF/AYgroelR (Mitrovic et al., 2011) specific for phytoplasma 16S rRNA and groEL gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from four symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The PCR productions were sequenced and the data were deposited in GenBank. The two gene fragments of the DNA extracted from the symptom plant samples were all identical, with the length of 1340 bp 16S rRNA (GenBank accession: MW353909) and 1312 bp groEL (MW353709) gene sequence fragments, putatively encoding 437 (groEL) amino acids sequence. The phytoplasma strain was named as Waltheria indica virescence (WiV) phytoplasma, WiV-hnda strain. A Blast search based on the 16S rRNA gene fragment of WiV-hnda phytoplasma strain revealed the highest level of sequence identities (99.85%) with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628) from Japan (Oshima et al., 2004); Periwinkle virescence phytoplasma strain PeV-hnhk (KP662136), Chinaberry witches'-broom phytoplasma strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), all the strains from Hainan island of China (Yu et al., 2017). A Blast search based on the groEL gene sequence fragment of WiV-hnda indicated 99.92% sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup) such as Onion yellows phytoplasma strain OY-M (AP006628). Homology and phylogenetic analysis by DNAMAN 5.0 and MEGA 7.0 software indicated that the phytoplasma strains of WiV-hnda, OY-M, PeV-hnhk, CWB-hnsy1 and CWB-hnsy2 were clustered into one clade based on the 16S rRNA gene fragments. WiV-hnda, OY-M and Aster yellow witches'-broom (AYWB) (CP000061) phytoplasma strains were clustered into one clade based on the groEL gene fragments. To our knowledge, this was the first time that Waltheria indica virescence disease induced by 16SrI-B subgroup phytoplasma strain was reported in China. Genetic analysis showed that WiV-hnda was closely related to the phytoplasma strains causing Onion yellows in Japan, Periwinkle virescence and Chinaberry witches'-broom disease in China.