A pot experiment was conducted to evaluate the efficacy of bacteriophage against tobacco BW disease and to determine the optimal dose. The pot experiment was carried out according to the method described by Le et al. (2020) and Eisfeld et al. (2022) with slight modifications. Tobacco seedlings were transplanted to a pot at the four-leaf stage, one seedling was used per pot and 500 g nursery substrate was added (Xiang Hui Agriculture Co., Ltd., China). After seedlings rooted, the experiments were grouped and performed based on Table 1; each group had three replications (18 seedlings per replication). BW pathogen R. ps. NdE (10⁵ CFU/mL * 20 mL per seedling) was inoculated with BW by non-invasive root irrigation. After the pathogen inoculated for 6 h and 72 h, the phage agents (10⁸ PFU/ml) with different dilutions (0, 10, 50, 100, and 200) were used with the same method (non-invasive root irrigation, 20 mL per seedling) followed by cultivation in a suitable climate. The temperature was 28 ± 3°C, humidity 60%–90%, 14 h light-10 h dark, and full spectrum 6,000 lm. Sterile water (about 25 mL/pot) was added to the pot every 2–5 days to maintain the humidity of the nursery substrate in pot at 25%–40%.

After 30 days of pathogen inoculation, the BW incidence rate, disease index, and relative control effect of phage agents were calculated according to the GB/T23222-2008 (Grade and investigation method of tobacco diseases and insect pests) (National public service platform for standards information, n.d.). The tobacco plant BW disease classification level was assessed on a scale of 0–9: 0 = no symptoms; 1 = half of the leaves on the diseased side were wilted or withered; 3 = black strip on stem, but not more than half of the stem height, or half to two-thirds of the diseased side leaves withered; 5 = the black strip on the stem exceeds half of the stem height, but does not reach the top of the stem, or more than two thirds of the leaves on the diseased side are wilted; 7 = the black spot on the stem reaches the top of the stem, or the leaves of the diseased plant are all withered; and 9 = a dead plant.

The BW incidence rate (IR) was the BW disease plants ratio; the disease index (DI) and control effect (CE) were calculated with Eqs 1, 2.

The calculation formula of DI and CE is as follows:

DI: disease index; n1–n5: the number of plants in each disease classification level; n: the number of plants investigated; CE: control effect (T versus C); C: disease index of control group; T: disease index of treatment group.

The experimental field is located in a tobacco field (28°13’ N, 112°32′ E) in Yuxin Village, Shuangfu Town, Ningxiang City, Hunan Province, China. The area has a subtropical humid monsoon climate; in the tobacco growing season (March to July), the average monthly temperature and average monthly precipitation were 21.6°C and 168 mm. The experimental site was 56 m above sea level, and the soil was brown soil with a pH value of 6.13, organic matter 17.84 g/kg, total nitrogen 1.29 g/kg, alkaline nitrogen 83.14 mg/kg, available phosphorus 113.91 mg/kg, and available potassium 254.36 mg/kg. The tobacco cultivar Yunyan 87 was tested. The tobacco seedlings were transplanted at the five-leaf seedling stage on March 20. The experiment was divided into three groups, namely the control group (FCK) without treatment, the General fungicide group (FZS), where a chemical fungicide – Zhongshengmycin (Yi et al., 2022) – was applied according to the recommended dosage, and the Phage treatment group (FPQ43W), where phage was used as 10⁸ PFU/ml-20 mL-plants. All the agents were applied twice by non-invasive root irrigation on 20 May and 20 June. The disease survey was carried out on July 10 (about a week that before the tail leaves were harvested) according to the method described in the pot experiment above.

The bacteria, as shown in Figure 1A, had red smooth colonies on TTC plates. The bacteria were isolated from tobacco BW disease stem with bacterial ooze by plate streaking in Hunan, China, and named NdE. A blastn analysis in NCBI showed that strain NdE had >99% similarity with the bacteria Ralstonia solanacearum species complex (Rssc) (Supplementary Table S1). A phylogenetic analysis showed that strain NdE was clustered into a cluster with Ralstonia pseudosolanacearum (NR_134148.1) and placed into Rssc clade (Supplementary Figure S1). Therefore, the bacteria NdE identified as Ralstonia pseudosolanacearum is a member of Rssc.

The phage was isolated from rhizosphere soil, which was collected from a field in Hunan Province, China with a high incidence of tobacco BW. The phage produced small and clear plaques with a diameter of approximately 1–2 mm after overnight incubation at 28°C on the top layer containing 0.75% agar of a double-layered NB plate using R. ps. NdE strain as a host (Figure 1B); the phage was named Ralstonia phage PQ43W. Transmission electron microscopy (TEM) was used to visualize the morphology and for classification (Figures 1C,D). The phage has a polygonal head with an average capsid diameter of 86 ± 4 nm (n = 10), a long tail with a length of 123 ± 3 nm and a width of 18 nm ± 3 nm (n = 10), and a baseplate and tail fibers of approximately 20 ± 2 nm (n = 10) in length (Figure 1B). Based on the morphology and structural features of the phage virions, it is typical for members of class Caudoviricetes, according to the standard of the International Committee on Taxonomy of Viruses (ICTV).

Genome sequencing showed that the phage PQ43W genome was dsDNA and circular, and the complete genomic DNA sequence (GenBank: PP405626) indicated that its genome was 47,156 bp long with guanine-cytosine content of 64.27%. The contents of G, C, A, and T were 32.45, 31.82, 17.73, and 18.00%, respectively. It has 65 predicted open reading frames (ORF1–ORF65); 39 ORFs were encoded on the plus strand and 26 ORFs were encoded on the minus strand (Figure 3; Supplementary Table S2). The total length of the ORFs sequence was 42,516 bp and the average length was 654 bp per ORFs. The coding percentage was 90.16%.

A total of 65 potential open reading frames (ORFs) were identified (Figure 3; Supplementary Table S2). Of them, seven had no significant similarity with any of the protein sequences in the searched databases and 36 were annotated as conserved, conserved hypothetical, or unnamed proteins with no assigned functions. Only 29 were predicted proteins that shared homology with other phages or bacteria with assigned functions based on KEGG analysis (Table 2). Of them, there were six phage-associated DNA replication and repair proteins, including RusA-like Holliday junction resolvase (ORF22), exonuclease (ORF24), DNA primase (ORF25), DNA helicase (ORF33), DNA polymerase (ORF34), and adenosine (5′)-pentaphospho-(5″)-adenosine pyrophosphohydrolase (ORF40). There were two phage-associated lysis proteins: holin (ORF16) and lysozyme (ORF17). There was one phage-associated packaging gene: terminase large subunit (ORF64). There was one phage-associated transcriptional regulator: phelix-turn-helix transcriptional regulator (ORF26). There were 19 phage-associated structural genes proteins, such as baseplate wedge subunit (ORF1) and virion structural protein (ORF2).

Figure 4 shows a blastn search using the nucleotide in NCBI Genbank that was homologous to the Burkholderia phage Bcep43 (NC_005342.2, 16% query coverage, and 76.55% identity) and Burkholderia phage BcepNY3 (NC_009604.1, 9% query coverage and 76.52% identity) with the highest score. It also shows the genome comparison analysis of Bcep43 and BcepNY3. The gene arrangement structure of PQ43W was significantly different from that of Bcep43 (Supplementary Table S3) and BcepNY3 (Supplementary Table S4). However, there were some similarities in some regional sequences (such as ORF1 ~ ORF15; ORF22 ~ ORF24; ORF25; ORF33 ~ ORF36; ORF40 ~ ORF65). Compared with Bcep43 or BcepNY3, the gene arrangement structure is very different, and the genes with the same function had different distribution positions and directions in the genome.

As further taxonomic information was not confirmed by the morphological identification, we resorted to genome-wide analyses at the protein level to clarify further taxonomic classification information with vContact3. The results in Table 3 show that PQ43W belongs to a novel genus of subfamily Kantovirinae (Table 3; Supplementary Table S5).

In addition, the classification with VICTOR using the “amino acid” option analysis to build phylogenetic trees was refined and constructed. Since the reference sequence of phylogenetic trees is directly related to the analysis result, it is very important to select appropriate, taxonomically close and representative sequences. So, in this study the reference sequence for VICTOR analysis were from five parts: (i) all the phages in subfamily Kantovirinae classification in ICTV Taxonomy Browser (https://ictv.global/taxonomy; two genera, five species, five phages); (ii) all the phages at subfamily Kantovirinae classification in NCBI taxonomy browser (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=2946634; two genera, five species, 13 of no rank, 18 phages); (iii) all the phages at subfamily Kantovirinae (Prediction) classification by vContact3 (database v220) (Supplementary Table S5; four genera, 12 phages.); (iv) Top 12 phages with the highest similarity in NCBI blastn with PQ43W genome-wide sequence (Supplementary Table S6; three genera, 12 phages); (iv) two Ralstonia phages were not in the subfamily Kantovirinae classification in ICTV and NCBI taxonomy database (Supplementary Table S6, Genus Cimandefvirus) and were used as outgroups. Subsequently, 32 phages were selected and listed in Supplementary Table S7 for further analysis.

The VICTOR analysis of phylogenomic GBDP trees inferred with the formulas D0, D4, and D6 were shown in Supplementary Figures S2–S4, respectively. Then, a summarized result based on the prediction taxonomy was shown in Supplementary Table S8. The prediction taxonomic of PQ43W showed the same results at the Phylum, Subfamily, and Genus classifications in formulas D0 and D6. PQ43W belongs to a novel Subfamily and novel Genus that does not have the same taxonomic phage at Subfamily and Genus taxonomic classification levels. In the formula of D4, the prediction taxonomic showed PQ43W with Ralstonia phage Eline (NC 054957.1) and Ralstonia phage Gerry (NC 054959.1) has the same taxonomic classification at Genus Cimandefvirus (Family: unknown; Subfamily: unknown). Considering the yielding average supports were 73, 69, and 76% at the formulas D0, D4, and D6, respectively, the prediction taxonomic results of PQ43W inferred in formulas D0 and D6 were more accurate than D4. In addition, eight phage predictions in Subfamily Kantovirinae in vContact3 also appeared in the prediction Family classification through VICTOR analysis with formulas D0 and D6, but the results analyzed with formula D4 was not. That also indicated the formulas D0 and D6 were more suitable for PQ43W by VICTOR analysis than the formula D4. So, PQ43W may belong to a novel Genus in Subfamily Kantovirinae.

A comparative analysis of genomic nucleotide similarity by VIRDIC was shown in Figure 5 and Supplementary Table S8 with 32 other related phages (Supplementary Table S7). The results of intergenomic similarity between PQ43W and these phages were 0.1–35.0% (Figure 5), and the species and genus clusters table is different from the 32 related phages (Supplementary Table S9). Based on the principle of classifying bacteriophage on genus in Bacterial and Archaeal Viruses Subcommittee (BAVS), a genus is a cohesive group of viruses sharing a high degree of nucleotide sequence similarity (>50%) that is distinct from viruses of other genera (Adriaenssens and Brister, 2017). So, PQ43W belongs to a novel genus in the class Caudoviricetes.

Therefore, PQ43W belongs to a novel genus of subfamily Kantovirinae under class Caudoviricetes.

To determine evolutional relationships between PQ43W and its 32 related phages (Supplementary Table S7), a phylogenetic tree based on the genome-wide nucleic acid sequence showed that PQ43 is adjacent to the genera of Naesvirus, Bakolyvirus, Beograduvirus, and Tsukubavirus, but distinct from them, in a separate clade (Figure 6A). In addition, the phylogenetic tree based on the three “signature gene products (tail fiber protein, portal protein, and terminase large subunit)” (Addy et al., 2019; Khan et al., 2021; Sasaki et al., 2021) of amino acid sequences showed that PQ43W is more closely related to the clade genera of Bakolyvirus (Figure 6B), Naesvirus (Figure 6C), or Aresaunavirus (Figure 6D), but is not in their clades. So, PQ43W has closer evolutional relationships to the genera Naesvirus, Bakolyvirus, Beograduvirus, Tsukubavirus, or Aresaunavirus in the phylogenetic analysis, though PQ43W belongs to a novel genus and was placed in a clade alone in Figure 6.

A pot experiment to address phage application for the control of tobacco BW disease caused by R. ps. NdE was conducted. An obvious control effect result on tobacco BW disease was surveyed and statistically analyzed after 30 days (Supplementary Figure S6; Table 4). It is easily observed in Supplementary Figure S6 that PQ43W could decrease the tobacco BW disease incidence rate (IR) and disease index (DI), and was consistent with the disease statistics result (Table 4). The PQ43W application groups showed a lower BW incidence rate (IR) and disease index (DI) to compared with the control group (CK+) in Table 4. On the whole, a higher titer of PQ43W application groups showed a better control effect on tobacco BW that the IR and DI indices were lower, especially the groups T1 and T10 had more than 50% CE on the control of tobacco BW for 30 days. What’s importantly, group T1 of the CE index increased to 92.80% for 30 days by root irrigation application at 10⁸ PFU/ml titers significantly.

Subsequently, a field experiment was conducted and the result showed in Table 5, based on the optimal dose of pot experiment (Table 4; T1: 10⁸ PFU/ml). Compared to the control treatment (FCK), PQ43W treatment (FPQ43W) had a lower Incidence Rate (IR) that was significantly reduced from 69.33% to 30.00% (p < 0.05). The Disease Index (DI) was also significantly lower and reduced from 17.19 to 3.78% (p < 0.05). In addition, the Control Effect (CE) of PQ43W treatment (FPQ43W: 78.02%) was significantly better than the traditional pesticide Zhongshengmycin treatment (FZS: 44.41%), (p < 0.05). At the same, the indexes of IR and DI in FPQ43W were lower than FZS, significantly (p < 0.05). Therefore, PQ43W of phage therapy to control BW in pot and field experiments indicated that PQ43W is a promising resource in controlling tobacco BW disease.