Waltheria indica Is a New Host of Phytoplasma Belonging to 16SrI-B Subgroup Associated with Virescence Symptoms in China

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 (Mitrović 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.

Molecular Identification and Characterization of Two Groups of Phytoplasma and Candidatus Liberibacter Asiaticus in Single or Mixed Infection of Citrus maxima on Hainan Island of China

“…Many plants could be infected by phytoplasmas belonging to different groups [ 27 , 29 , 30 ]. Trema tomentosa , showing witches’ broom disease, was infected by the phytoplasma strains belonging to the 16SrXXXII-D subgroup [ 29 ]; Melochia corchorifolia witches’ broom disease was caused by the phytoplasma strains belonging to the 16SrII group [ 27 ]; Waltheria indica virescence disease was associated with the 16SrI group phytoplasma strains [ 30 ]; areca palm yellow leaf disease was also induced by the phytoplasmas belonging to the 16SrI group [ 38 , 39 ]. Citrus Huanglongbing disease symptoms in citrus plants, such as Citrus maxima , Citrus sinensis and Citrus × limon , were also found on the island and were identified to be associated with the pathogen of Candidatus Liberibacter asiaticus [ 23 ].…”

“…Economic palm crops, such as areca palm, coconut and oil palm, were also planted on the farm. Moreover, there were many phytoplasma-infected plants reported previously, such as Melochia corchorifolia , Emilia sonchifolia , Trema tomentosa and Waltheria indica , which were also found on the farm [ 27 , 28 , 29 , 30 ]. Total DNA of the diseased plant samples was extracted based on the CTAB method, according to the rapid extraction kit for plant genomic DNA (Tiangen Biotech Co., Ltd., Beijing, China) [ 31 ].…”

“…The disease symptoms of Citrus maxima are similar to the symptoms caused by Citrus Huanglongbing disease. Furthermore, some phytoplasma diseases were reported previously in the regions associated with the disease symptoms of Citrus maxima [ 27 , 28 , 29 , 30 ]. Therefore, the plants with disease symptoms were suspected to be associated with the pathogens of phytoplasma and Candidatus Liberibacter asiaticus, based on a field investigation and a previous study [ 17 , 18 , 19 , 23 , 27 , 28 , 29 , 30 ].…”

“…Furthermore, some phytoplasma diseases were reported previously in the regions associated with the disease symptoms of Citrus maxima [ 27 , 28 , 29 , 30 ]. Therefore, the plants with disease symptoms were suspected to be associated with the pathogens of phytoplasma and Candidatus Liberibacter asiaticus, based on a field investigation and a previous study [ 17 , 18 , 19 , 23 , 27 , 28 , 29 , 30 ]. At present, there are no reports of mixed infection with phytoplasma and Candidatus Liberibacter asiaticus on the citrus plant hosts and even other plant hosts on Hainan Island in China.…”

Insight into the protein degradation during the broad bean fermentation process

“…The sequencing results were submitted to GenBank for similarity analysis. Finally, the phylogenetic tree was established using MEGA7.0 (Yu et al., 2021 ).…”