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Bischofia polycarpa (Levl.) Airy Shaw witches'-broom disease is widespread in southern China. Infected trees show symptoms of severely proliferating shoots, reduced leaf size, yellowing and reddening of leaves, and internode shortening. We detected the phytoplasma in diseased tissues by 4′,6-diamidino-2-phenylindole-HCl fluorescence microscopy and polymerase chain reaction using the universal primers P1/ P7. We conducted sequence similarity analyses based on 16S rRNA and 23S rRNA genes, the spacer region between the 16S and 23S rRNA genes (ISR), the partial ribosomal proteinencoding operon (rp), and the elongation factor Tu (tuf) gene of the BiWB phytoplasma. These analyses unanimously showed that the BiWB phytoplasma belongs to the elm yellows group (the 16SrV group). Phylogenetic and computersimulated restriction fragment length polymorphism analyses using the nearly full-length 16S rRNA gene sequence demonstrated that the BiWB phytoplasma is more closely related to the subgroup 16SrV-B than to the other subgroups in 16SrV. It may represent a novel subgroup, designed as 16SrV-H, which is distinct from other subgroups of phytoplasmas in the 16SrV group.

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.

Emilia sonchifolia is a medical plant belonging to the family of Asteraceae, mainly used as a traditional Chinese medicine with the function of anti-inflammatory, analgesic, antibacterial and so on. During October to November 2020, the plants showing abnormal symptoms including witches’-broom, internode shortening, leaf chlorosis and leaflet were found in Hainan province, a tropical island of China. The total DNA of the plant samples were extracted using 0.10 g fresh plant leaves using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 and secAfor1/secArev3 specific for phytoplasma 16S rRNA and secA gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from three symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The two gene fragments of the DNA extracted from the symptomatic plant samples were all identical, with the length of 1324 bp 16S rRNA and 760 bp secA gene sequence fragments, putatively encoding 253 (secA) amino acids sequence. The phytoplasma strain was named as Emilia sonchifolia witches’-broom (EsWB) phytoplasma, EsWB-hnda strain. To our knowledge, this was the first report that Emilia sonchifolia witches’-broom disease was caused by the phytoplasma belonging to16SrII-V subgroup in Hainan island of China, with close relationship to 16SrII peanut witches’-broom group phytoplasma strains infecting the plants like peanut, Desmodium ovalifolium and cleome from the same island of China and cassava from Viet Nam.

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.

Studies on the genetic diversity, key gene regulation and control of phytoplasma, which cause many diseases with various host plants and have a wide geographical distribution, will be conducive to facilitating integrated disease control. The large DNA fragments including tuf gene sequences and upstream six genes from PaWB-sdyz, PaWB-fjfz and LY-fjya1 strains were amplified using long fragment PCR primers. Sequence characteristic of conserved regions of the phytoplasma gene promoter and MLSA were performed. Upstream sequences adjoining the tuf gene was recombined with promoter-probe vector pSUPV4 to analyze their promoter activity. The sequences, 12,745-12,748 bp in length, of upstream tuf genes were amplified from the three strains. Comparative analysis showed that the gene structure order of the tuf gene and its upstream six gene sequences of PaWB-sdyz, PaWB-fjfz, LY-fjya1, OY-M, AYWB, PAa, SLY, AT phytoplasma strains were identical in the arrangement of 5'-rplL-rpoB-rpoC-rps12-rps7-fusA-tuf-3'. The potential sequence pattern of conserved region of the phytoplasma promoter was deduced: T 90 T 100 G 92 T 75 G 67 A 85 (-35 region); T 90 A 96 T 92 A 98 T 73 T 90 (-10 region). The different phytoplasma strains were clearly divided with comparatively high bootstrap values based on MLSA of coding genes, non-coding sequences, and deduced amino acid sequences of rplL-tuf nucleotide sequences. Genetic variation was comparatively high in the non-coding nucleotide sequences. A 130-bp upstream sequence of the tuf gene in PaWB-fjfz, LY-fjya1 strains and a