Xanthomonas bacteriophages: a review of their biology and biocontrol applications in agriculture.

Phytopathogenic bacteria are economically important because they affect crop yields and threaten the livelihoods of farmers worldwide. The genus Xanthomonas is particularly significant because it is associated with some plant diseases that cause tremendous loss in yields of globally essential crops. Current management practices are ineffective, unsustainable and harmful to natural ecosystems. Bacteriophage (phage) biocontrol for plant disease management has been of particular interest from the early nineteenth century to date. Xanthomonas phage research for plant disease management continues to demonstrate promising results under laboratory and field conditions. AgriPhage has developed phage products for the control of Xanthomonas campestris pv. vesicatoria and Xanthomonas citri subsp. citri. These are causative agents for tomato, pepper spot and speck disease as well as citrus canker disease.Phage-mediated biocontrol is becoming a viable option because phages occur naturally and are safe for disease control and management. Thorough knowledge of biological characteristics of Xanthomonas phages is vital for developing effective biocontrol products. This review covers Xanthomonas phage research highlighting aspects of their ecology, biology and biocontrol applications.

Background

The genus Xanthomonas; is a well-studied group of plant-associated Gram-negative bacteria that belong to the family Xanthomonadaceae subclass Gammaproteobacteria [1]. An estimated 27 species is pathogenic to approximately 400 plants. These include but not limited to sugar cane, beans, cassava, cabbage, banana, citrus, tomatoes, pepper and rice [2]. The life cycle of Xanthomonas has two stages: epiphytic and endophytic [3]. The epiphytic stage starts once bacteria colonize the surfaces of a new plant using adhesion ligands such as bacteria surface polysaccharides [4], adhesion proteins [5], and type IV pili [6]. After colonization comes biofilm formation, which then protects the bacteria from environmental stress factors [7]. The endophytic stage is characterised by bacterial entry into plant tissue via lesions or stomata and eventual movement throughout the vascular system. The bacteria re-emerge onto the plant surfaces once their population reaches the threshold, transmission occurs to new hosts and the infection cycle repeats [3].

Although Xanthomonas species are well-studied, the genus remains responsible for many crop diseases that cause crop yield losses in economically important crops worldwide [2, 3].

The current management methods used to control Xanthomonas-associated diseases include de-budding, uprooting, burying and burning of infected plant tissues, sterilization of garden tools, and application of copper-based pesticides and antibiotics such as streptomycin [8-10]. The concerns raised about ineffective cultural practices, copper-based pesticide, antibiotic resistance problems, and environmental chemical contamination have piqued worldwide interest in Xanthomonas phage research and biocontrol application in agriculture.

Phages are viruses that infect and replicate in bacteria. Phage replication cycles include temperate and lytic pathways with the lytic pathway being the easier and more important pathway for employment in phage biocontrol. In the lytic pathway the phages bind to the surface of bacteria after which they inject their DNA and replicate inside the cell. This results in the production of phage progeny that lyse and kill the bacteria [11]. In the temperate pathway, once the phage has successfully bound and injected its DNA into the host, the phage may either stably integrate into the genome of the bacteria or enter into the lytic life cycle. Using temperate phages in phage biocontrol poses some disadvantages in that, once the phage inserts its genome into the bacterial DNA chromosome, the prophage is transmitted to daughter cells by horizontal gene transfer thereby providing undesirable genes that may aggravate bacterial disease, e.g. filamentous phage CTX Φ that encodes cholera toxin [12].

Historically, bacteriophage-based biocontrol specific for phytopathogen Xanthomonas dates back to the early nineteenth century, when a filtrate of decomposing cabbage stopped the spread of cabbage-rot disease caused by Xanthomonas campestris pv. campestris, [13]. Decades later, similar biocontrol success was reported with phage-containing lysates that inhibited bacterial spot disease in peach caused by Xanthomonas campestris pv. pruni [14, 15]. A number of phage applications have progressed from in-vitro experiments to field trials. These include studies on bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria [16]; geranium bacterial blight caused by Xanthomonas campestris pv. pelargonii [17]; leaf blight of onion caused by Xanthomonas axonopodis pv. allii [18]; citrus canker and citrus bacterial spot caused by Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. citrumelo [19]; asiatic citrus canker caused by Xanthomonas axonopodis pv. citri [20] and Xanthomonas citri subsp. citri [21]; bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae [22, 23] and bacterial leaf blight of welsh onions caused by Xanthomonas axonopodis pv. allii [24]. Two Xanthomonas phage products manufactured by AgriPhage [25] have been shown to successfully control pathogens that cause tomato and pepper spot disease and citrus canker disease.

Owing to the growing interest in using Xanthomonas phages to control the genus Xanthomonas, this review emphasizes the taxonomy, ecology, biology and biocontrol applications.

Main text

Taxonomy of Xanthomonas phages

A total of 168 Xanthomonas phages described to date classify into orders: Caudovirales with 151 phages and Tubulavirales with 17 phages (Additional file 1). According to the International Committee on Taxonomy of Viruses (ICTV), Caudovirales contain 9 families [26] and Xanthomonas phages reported in literature or National Centre for Biotechnology Information (NCBI) database belong to 5 families namely: Podoviridae, Siphoviridae, Myoviridae, Autographiviridae, and Herelleviridae (Additional file 1). A total of 71 Xanthomonas phages belong to Myoviridae, 42 belong to Podoviridae, 34 belong to Siphoviridae, 17 belong to Inoviridae, 3 belong to Autographiviridae and 1 member to Herelleviridae. Order Caudovirales possess tubular tails that can be either long and contractile (Myoviridae), long and non-contractile (Siphoviridae), or short and non-contractile (Podoviridae, Autographiviridae) [26-28]. The capsids of Caudovirales are non-enveloped, exhibit icosahedral symmetry with a typical diameter of 45 and 170 nm and encapsidate linear double-stranded genomes. Their genome length is between 39,980 and 384,670 nucleotides, carries between 40 and 592 open reading frames and has a guanine-cytosine (GC) content between 40 and 66% (Additional file 1). On the other hand, Tubulavirales consist of one family; Inoviridae. They are filamentous virions that possess helical symmetry and non-enveloped capsid (Additional file 1). The inovirus genomes are small, circular, single-stranded DNA molecules that range between 6000 and 8500 nucleotides. The genome encodes between 9 and 14 open reading frames and has a GC content between 57 and 60% (Additional file 1).

Ecology and host range

Ecology: geographical distribution, environmental isolation source, host bacteria and plant disease.

Geographical distribution

The geographical distribution of Xanthomonas phages spans parts of Asia, North America, South America, Europe, Zealandia and North Africa. The countries where the phages are isolated are summarized in Table 1. The Xathomonas phages are distributed across the world depending on the pathogen that is present in that part of the world.

Table 1

Country of isolation of Xanthomonas phages, their families and host strain/s they infect

Country of isolation	Xanthomonas phage/s	Family	Causative bacterium	Reference
China	Xop41	Siphoviridae	X. oryzae pv. oryzae	[29]
China	Xoo-sp1,Xoo-sp2, Xoo-sp3, Xoo-sp4, Xoo-sp5, Xoo-sp6, Xoo-sp7, Xoo-sp8, Xoo-sp9, Xoo-sp10, Xoo-sp11, Xoo-sp12, Xoo-sp13, Xoo-sp14, Xoo-sp15	Siphoviridae	X. oryzae pv. oryzae	[30]
China	X1, X2, X3, X4, X5	Myoviridae	X. oryzae pv. oryzae	[31]
China	Xoo-sp14	Myoviridae	X. oryzae pv. oryzae	[32]
China	Xoo-sp13	Myoviridae	X. oryzae pv. oryzae	[33]
China	Xf409	Inoviridae	X. oryzae pv. oryzicola	[34]
Taiwan	Xp10, Xp12, Xp20	Siphoviridae	X. oryzae pv. oryzae	[35]
Taiwan	ϕXc10	Autographiviridae	X. citri pv. glycines, X. campestris pv. campestris, X. campestris pv. citri	[36]
Korea	P8L, P27L, P30L, P59L, P73L	Siphoviridae	X. oryzae pv. oryzae	[22]
Korea	P4L, P4M, P6M, P6M1, P14M, P14M1, P18M, P23M1,P33M, P37L, P37M, P37M1, P41M, P43M, P45M, P47M, P50M, P53M, P54M, P57M, P58M, P60M, P61M, P62M, P66M, P68M, P70M, P71L, P72M	Myoviridae	X. oryzae pv. oryzae	[22]
Japan	XacN1	Myoviridae	X. citri	[37]
Viet Nam	Phage Xaa_vB_ϕ31	Autographiviridae	X. euvesicatoria pv. allii XaaBL11	[38]
Philippines	XPP1	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP2	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP3	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP4	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP6	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP8	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPP9	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPV1	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPV2	Myoviridae	X. oryzae pv. oryzae	[39]
Philippines	XPV3	Myoviridae	X. oryzae pv. oryzae	[39]
India	φXOF1	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOF2	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOF3	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOF4	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOT1	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOT2	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOM1	Siphoviridae	X. oryzae pv. oryzae	[23]
India	φXOM2	Siphoviridae	X. oryzae pv. oryzae	[23]
India	Xcc9SH3	Siphoviridae	X. campestris pv. campestris	[40]
India	Xcc3SH, Xcc6SH3, Xcc7SH3, Xcc8SH3, Xcc9SH3, Xcc14SH3, JPS-xcc-3_P1, JPS-xcc-4_P1, JPS-xcc-7_P1, NBL-xcc-7_P1, NBL-xcc-4_P1, NBL-xcc-7_P1, NBL-xcc-3_P1, NBL-xcc-9_P1,NFS-xcc-9_P1, GRW-xcc-9_P1, NFS-xcc-9_P2, NBL-xcc-9_P2, GRW-xcc-10_P1, NFS-xcc-10_P1, NBL-xcc-10_P1, GRW-xcc-14_P1, NFS-xcc-14_P1, NBL-xcc-14_P1, GRW-xcc-17_P1, NFS-xcc-17_P1, NBL-xcc-17_P1, GRW-xcc-19_P1, NFS-xcc-19_P1, NBL-xcc-19_P1	n/a	X. campestris pv. campestris	[40]
India	Xap-1, Xap-2, Xap-3, Xap-4, Xap-5	n/a	X. axonopodis pv. punicae	[41]
USA	T7-like podophage Pagan	Autographiviridae	Xanthomonas sp., rice isolate ATCC PTA-13101	[42]
USA	Cf2	Inoviridae	X. citri pv. citri	[43]
USA	Phage River Rider	Podoviridae	X. fragariae	[44]
Mexico	Xaf13	Inoviridae	X. vesicatoria	[45]
Mexico	ϕXaf18	Myoviridae	X. vesicatoria	[46]
Brazil	XC2	Myoviridae	X. campestris pv. campestris	[47]
Chile	f30-Xaj	Podoviridae	X. arboricola pv. juglandis	[48]
Chile	f20-Xaj	Podoviridae	X. arboricola pv. juglandis	[48]
Russia	DB 1	Siphoviridae	X. campestris pv. campestris	[49]
Serbia	Kɸ1, Kɸ15	Myoviridae	X. euvesicatoria	[50]
Serbia	Kɸ1, Kɸ2, Kɸ3, Kɸ4, Kɸ5, Kɸ6, Kɸ7, Kɸ8, Kɸ9, Kɸ15	n/a	X. euvesicatoria	[50]
New Zealand	BP60C1–3, Bp10, Bp20, Bp22	Myoviridae	X. campestris pv. juglandis	[51]
New Zealand	P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26	Siphoviridae	X. arboricola pv. juglandis	[52]
France	Phage Olaya	Podoviridae	X. albilineans CFBP2523	[53]
France	Phage Bolivar	Podoviridae	X. albilineans CFBP2523	[54]
France	Phage Usaquen	Podoviridae	X. albilineans CFBP2523	[55]
France	Phage Alcala	Podoviridae	X. albilineans CFBP2523	[56]
France	Phage Fontebon	Podoviridae	X. albilineans CFBP2523	[57]
France	Phage Soumapaz	Podoviridae	X. albilineans CFBP2523	[58]
Belgium	FoX7	Myoviridae	X. campestris pv. campestris GBBC 1412	[59]
Belgium	FoX6	Myoviridae	X. campestris pv. campestris GBBC 1412	[60]
Belgium	FoX5	Myoviridae	X. campestris pv. campestris GBBC 1419	[61]
Belgium	FoX3	Myoviridae	X. campestris pv. campestris GBBC 1420	[62]
Belgium	FoX2	Myoviridae	X. campestris pv. campestris GBBC 1419	[63]
Belgium	FoX1	Myoviridae	X. campestris pv. campestris GBBC 1419	[64]
Belgium	FoX4	Siphoviridae	X. campestris pv. campestris GBBC 1412	[65]
Moldova	Phage PPDBI	Podoviridae	X. campestris pv. campestris	[49]
Egypt	Phage 1, Phage 2	n/a	X. axonopodis	[66]

n/a not available; X Xanthomonas; pv pathovar; sp species

Ecology: environmental isolation source, host bacteria and plant disease

The environmental isolation source of Xanthomonas phages as well as bacterial host and plant disease are summarized in Table 2. These viruses establish infection in Xanthomonas pathovars responsible for a range of plant diseases including but not limited to bacterial leaf blight, black rot, bacterial leaf spot and citrus canker (Table 2). The majority of Xanthomonas phages are isolated from infected plant phyllosphere and rhizosphere, while others are isolated from compost, sewage and water (irrigation, pond, freshwater lakes and rivers) (Table 2).

Table 2

Ecology of selected Xanthomonas phages: environmental source of isolation, host bacteria and plant disease

Xanthomonas phage/s	Environmental source	Host bacterium	Plant disease	Plant	Reference
Xop411	Xoo infected leaves	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[29]
Xp12	Xoo infected paddy water	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[67]
P4L, P4M, P6M, P6M1, P14M, P14M1, P18M, P23M1,P33M, P37L, P37M, P37M1, P41M, P43M, P45M, P47M, P50M, P53M, P54M, P57M, P58M, P60M, P61M, P62M, P66M, P68M, P70M, P71L, P72M, P8L, P27L, P30L, P59L, P73L	Xoo infected paddy water	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[22]
XPP1-XPP9, XPV1-XPV3	Xoo infected paddy water & soil	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[39]
X1, X2, X3, X4, X5	Xoo infected leaves	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[31]
φXOF1-φXOF4, φXOT1- φXOT2, φXOM1-φXOM2	Xoo infected leaves	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[23]
Xoo-sp1, Xoo-sp2, Xoo-sp3, Xoo-sp4, Xoo-sp5, Xoo-sp6, Xoo-sp7, Xoo-sp8, Xoo-sp9, Xoo-sp10, Xoo-sp11, Xoo-sp12, Xoo-sp13, Xoo-sp14, Xoo-sp15	Xoo infected paddy soil	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[30]
Xf	Xoo infected leaves	X. oryzae pv. oryzae	Bacterial leaf blight	Rice	[68]
Xcc3SH, Xcc6SH3, Xcc7SH3, Xcc8SH3, Xcc9SH3, Xcc14SH3, JPS-xcc-3_P1, JPS-xcc-4_P1, JPS-xcc-7_P1, NBL-xcc-7_P1, NBL-xcc-4_P1, NBL-xcc-7_P1, NBL-xcc-3_P1, NBL-xcc-9_P1,NFS-xcc-9_P1, GRW-xcc-9_P1, NFS-xcc-9_P2, NBL-xcc-9_P2, GRW-xcc-10_P1, NFS-xcc-10_P1, NBL-xcc-10_P1, GRW-xcc-14_P1, NFS-xcc-14_P1, NBL-xcc-14_P1, GRW-xcc-17_P1, NFS-xcc-17_P1, NBL-xcc-17_P1, GRW-xcc-19_P1, NFS-xcc-19_P1, NBL-xcc-19_P1	Xcc infected soil and leaves, river water	X. campestris pv. campestris	Black rot	Crucifers; cabbage, cauliflower, brasicca	[40]
Pg125	Xcc infected swede seed, compost & sewage	X. campestris pv. campestris	Black rot	Crucifers; turnip, cabbage, swede	[69]
Xcc φ1	Xcc infected soil	X. campestris pv. campestris	Black rot	Crucifers;broccoli, cabbage, cauliflower, radish	[70]
XTP1	Xcc infected soil	X. campestris pv. campestris	Black rot	Crucifers; cabbage	[71]
XcaP1	Xcc infected leaves	X. campestris pv. campestris	Black rot	Crucifers; cabbage	[72]
XC2	Xcc infected leaves	X. campestris pv. campestris	Black rot	Crucifer; cauliflower	[47]
DB 1	Xcc infected soil	X. campestris pv. campestris	Black rot	Crucifer, cabbage	[49]
XcuP3	Xcu infected fruit	X. campestris pv. cucurbitae	Bacterial leaf spot	Pumpkin	[72]
XcuP1	Xcu infected leaves	X. campestris pv. cucurbitae	Bacterial leaf spot	Zucchini	[72]
XholP1	Xho infected Leaves	X. campestris pv. holcicola	Bacterial leaf streak	Sorghum	[72]
Xp3-I	Xp infected soil	X. pruni	Bacterial leaf spot	Peach	[15]
Xp3-A.	Xp infected soil	X. pruni	Bacterial leaf spot	Peach	[15]
XprP1	Xpr infected stem	X. campestris pv. pruni	Bacterial leaf spot	Plum	[72]
XmaP1	Xma infected leaves	X. campestris pv. malvacearum	Bacterial blight	Cotton	[72]
XveP1	Xve infected leaves	X. campestris pv. vesicatoria	Bacterial leaf spot	Goosberry	[72]
Kɸ1, Kɸ2, Kɸ3, Kɸ4, Kɸ5, Kɸ6, Kɸ7, Kɸ8, Kɸ9, Kɸ15	Xeu infected leaves, stems, fruits, soil, seeds & irrigation water	X. euvesicatoria	Bacterial leaf spot	Pepper	[50]
Phages I to XX	Xtr infected grains	X. trifolii	Wheat disease	Wheat	[73]
X. phage 1 & X. phage 2	Xax infected leaves	X. axonopodis	Bacterial leaf spot	Pepper	[66]
Xap-1, Xap-2, Xap-3, Xap-4, Xap-5	Pond water	X. axonopodis pv. punicae	Bacterial leaf blight	Pomegranate	[41]
1, 20, 22, ΦPS, ΦSD, ΦSL, ΦRS, Φ56, Φ112, Pg60	Sewage, compost, Xp infected soil, seed & dry bean straw	X. phaseoli	Common blight of beans	Beans	[74]
Pg176, Pg177, Pg181,	Xp infected soil	X. phaseoli	Common blight of beans	Beans	[75]
Xanthomonas Siphophage Samson	Sewage	X. sp. strain ATCC PTA-13101	Bacterial leaf blight	Rice	[76]
Xanthomonas phage pagan	Fresh water	X. sp. strain ATCC PTA-13101	Bacterial leaf blight	Rice	[42]
Xanthomonas phage XacN1	Xci infected soil	X. citri	Asian citrus canker	Orange	[37]
BP60C1–3, Bp10, Bp20, Bp22	Xcj infected soil	X. campestris pv. juglandis	Walnut blight	Walnut	[51]
P1-P26	Xaj infected soil, leaves & fruit	X. arboricola pv. juglandis	Walnut blight	Walnut	[52]
XaF13	Xve infected soil	X. vesicatoria	Bacterial leaf spot	Pepper	[45]

X, Xanthomonas; pv, Pathovar; sp., species; Xanthomonas oryzae pv. oryzae; Xcc, Xanthomonas campestris pv. campestris; Xcu, Xanthomonas campestris pv. cucurbitae; Xho, Xanthomonas campestris pv. holcicola; Xp, Xanthomonas pruni; Xpr, Xanthomonas campestris pv. pruni; Xma, Xanthomonas campestris pv. malvacearum; Xve, Xanthomonas campestris pv. vesicatoria; Xeu, Xanthomonas euvesicatoria; Xtr, Xanthomonas trifolii; Xax, Xanthomonas axonopodis; Xp, Xanthomonas phaseoli; Xci, Xanthomonas citri; Xcj, Xanthomonas campestris pv. juglandis; Xaj, Xanthomonas arboricola pv. juglandis; Xve, Xanthomonas vesicatoria

Host range

Phages with a narrow host range infect one or few of the same bacteria strains, broad host range phages infect multiple strains of the same bacteria, and polyvalent phages infect several species or unrelated genera [77, 78]. A total of 148 Xanthomonas phages described in literature have a narrow, broad or polyvalent host range. Of these 52 have a narrow and 88 have a broad host range. The remaining 8 have a polyvalent host range. The lytic activity of phages with a narrow host range is between 13 and 57% while those with a broad range is between 60 and 100% (Table 3).

Table 3

Host range of Xanthomonas phages

Host range	Phage	Bacteria strain used	Number bacteria strains	Lysed bacteria strains	% lytic activity	Reference
Narrow	X. vesicatoria phage (chilli derived)	X. vesicatoria	8	4	50	[79]
Narrow	X. vesicatoria phage (datura derived)	X. vesicatoria	8	1	13	[79]
Narrow	XC2	X. campestris pv. campestris	10	5	50	[47]
Broad	Xoo-sp1	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp2	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp3	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp4	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp5	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp6	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp7	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp8	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp9	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp10	X. oryzae pv. oryzae	10	9	90	[30]
zBroad	Xoo-sp11	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp12	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp13	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp14	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Xoo-sp15	X. oryzae pv. oryzae	10	9	90	[30]
Broad	Kϕ1	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ2	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ3	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ4	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ5	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ6	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ7	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ8	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ9	X. euvesicatoria	59	59	100	[50]
Broad	Kϕ15	X. euvesicatoria	59	47	80	[50]
Broad	Xma-P1	X. pv. malvacearum	8	8	100	[72]
Broad	Xho-P1	X. campestris pv. holcicola	4	4	100	[72]
Broad	Xpr-P1	X. campestris pv. pruni	6	6	100	[72]
Broad	OP2	X. oryzae pv. oryzae	82	78	95	[80]
Broad	OP1h2	X. oryzae pv. oryzae	82	75	91	[80]
Narrow	OP1	X. oryzae pv. oryzae	82	46	56	[80]
Narrow	OP1h	X. oryzae pv. oryzae	82	20	24	[80]
Broad	φXOF1	X. oryzae pv. oryzae	6	4	67	[23]
Broad	φXOF2	X. oryzae pv. oryzae	6	4	67	[23]
Broad	φXOF3	X. oryzae pv. oryzae	6	5	83	[23]
Broad	φXOF4	X. oryzae pv. oryzae	6	6	100	[23]
Narrow	φXOT1	X. oryzae pv. oryzae	6	3	50	[23]
Narrow	φXOT2	X. oryzae pv. oryzae	6	3	50	[23]
Narrow	φXOM1	X. oryzae pv. oryzae	6	3	50	[23]
Narrow	φXOM2	X. oryzae pv. oryzae	6	3	50	[23]
Broad	X1	X. oryzae pv. oryzae	23	15	65	[31]
Broad	X2	X. oryzae pv. oryzae	23	21	91	[31]
Broad	X3	X. oryzae pv. oryzae	23	22	96	[31]
Broad	X4	X. oryzae pv. oryzae	23	21	91	[31]
Broad	X5	X. oryzae pv. oryzae	23	14	61	[31]
Broad	P4L	X. oryzae pv. oryzae	47	33	70	[22]
Broad	P4M	X. oryzae pv. oryzae	47	46	98	[22]
Broad	P6M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P6M1	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P8L	X. oryzae pv. oryzae	47	36	77	[22]
Broad	P14M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P14M1	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P18M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P23M1	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P27L	X. oryzae pv. oryzae	47	33	70	[22]
Broad	P30L	X. oryzae pv. oryzae	47	31	66	[22]
Broad	P33M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P37L	X. oryzae pv. oryzae	47	33	70	[22]
Broad	P37M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P37M1	X. oryzae pv. oryzae	47	46	98	[22]
Broad	P41M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P43M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P45M	X. oryzae pv. oryzae	47	33	70	[22]
Broad	P47M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P50M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P53M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P54M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P57M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P58M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P59L	X. oryzae pv. oryzae	47	31	66	[22]
Broad	P60M	X. oryzae pv. oryzae	47	28	60	[22]
Broad	P61M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P62M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P66M	X. oryzae pv. oryzae	47	46	98	[22]
Broad	P68M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P70M	X. oryzae pv. oryzae	47	47	100	[22]
Narrow	P71L	X. oryzae pv. oryzae	47	27	57	[22]
Broad	P72M	X. oryzae pv. oryzae	47	47	100	[22]
Broad	P73L	X. oryzae pv. oryzae	47	46	98	[22]
Narrow	Xcc3SH	X. campestris pv. campestris	17	6	35	[40]
Narrow	Xcc7SH	X. campestris pv. campestris	17	5	29	[40]
Narrow	Xcc6SH	X. campestris pv. campestris	17	7	41	[40]
Narrow	Xcc8SH	X. campestris pv. campestris	17	4	24	[40]
Narrow	Xcc9LK	X. campestris pv. campestris	17	5	29	[40]
Broad	Xcc9SH3	X. campestris pv. campestris	17	17	100	[40]
Narrow	Xcc14SH	X. campestris pv. campestris	17	7	41	[40]
Narrow	JPS-xcc-3_P1	X. campestris pv. campestris	17	6	35	[40]
Narrow	JPS-xcc-4_P1	X. campestris pv. campestris	17	6	35	[40]
Narrow	JPS-xcc-7_P1	X. campestris pv. campestris	17	6	35	[40]
Narrow	NBL-xcc-7_P1	X. campestris pv. campestris	17	6	35	[40]
Narrow	NBL-xcc-4_P1	X. campestris pv. campestris	17	4	24	[40]
Narrow	NBL-xcc-7_P1	X. campestris pv. campestris	17	5	29	[40]
Narrow	NBL-xcc-3_P1	X. campestris pv. campestris	17	3	18	[40]
Narrow	NBL-xcc-9_P1	X. campestris pv. campestris	17	8	47	[40]
Narrow	NFS-xcc-9_P1	X. campestris pv. campestris	17	6	35	[40]
Narrow	GRW-xcc-9_P1	X. campestris pv. campestris	17	3	18	[40]
Narrow	NFS-xcc-9_P2	X. campestris pv. campestris	17	5	29	[40]
Narrow	NBL-xcc-9_P2	X. campestris pv. campestris	17	7	41	[40]
Narrow	GRW-xcc-10_P1	X. campestris pv. campestris	17	7	41	[40]
Narrow	NFS-xcc-10_P1	X. campestris pv. campestris	17	3	18	[40]
Narrow	NBL-xcc-10_P1	X. campestris pv. campestris	17	5	29	[40]
Narrow	GRW-xcc-14_P1	X. campestris pv. campestris	17	8	47	[40]
Narrow	NFS-xcc-14_P1	X. campestris pv. campestris	17	12	71	[40]
Narrow	NBL-xcc-14_P1	X. campestris pv. campestris	17	7	41	[40]
Narrow	GRW-xcc-17_P1	X. campestris pv. campestris	17	9	53	[40]
Narrow	NFS-xcc-17_P1	X. campestris pv. campestris	17	3	18	[40]
Narrow	NBL-xcc-17_P1	X. campestris pv. campestris	17	5	29	[40]
Narrow	GRW-xcc-19_P1	X. campestris pv. campestris	17	8	47	[40]
Narrow	NFS-xcc-19_P1	X. campestris pv. campestris	17	12	71	[40]
Narrow	NBL-xcc-19_P1	X. campestris pv. campestris	17	7	41	[40]
Broad	Pg60	X. phaseoli	16	15	94	[69]
Broad	Pg176	X. phaseoli	16	14	88	[69]
Narrow	Pg177	X. phaseoli	16	7	44	[69]
Narrow	Pg181	X. phaseoli	16	9	56	[69]
Broad	P1	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P2	X. arboricora pv. juglandis	16	13	81	[52]
Broad	P3	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P4	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P5	X. arboricora pv. juglandis	16	13	81	[52]
Broad	P6	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P7	X. arboricora pv. juglandis	16	10	63	[52]
Broad	P8	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P9	X. arboricora pv. juglandis	16	11	69	[52]
Broad	P10	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P11	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P12	X. arboricora pv. juglandis	16	11	69	[52]
Broad	P13	X. arboricora pv. juglandis	16	11	69	[52]
Broad	P14	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P15	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P16	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P17	X. arboricora pv. juglandis	16	12	75	[52]
Broad	P18	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P19	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P20	X. arboricora pv. juglandis	16	14	88	[52]
Broad	P21	X. arboricora pv. juglandis	16	11	69	[52]
Broad	P22	X. arboricora pv. juglandis	16	12	75	[52]
Narrow	P23	X. arboricora pv. juglandis	16	5	31	[52]
Narrow	P24	X. arboricora pv. juglandis	16	5	31	[52]
Narrow	P25	X. arboricora pv. juglandis	16	7	44	[52]
Narrow	P26	X. arboricora pv. juglandis	16	5	31	[52]
Narrow	ϕ5A	X. axonopodis pv. allii	12	5	42	[24]
Narrow	ϕ5B	X. axonopodis pv. allii	12	5	42	[24]
Broad	ϕ6	X. axonopodis pv. allii	12	9	75	[24]
Narrow	ϕ7A	X. axonopodis pv. allii	12	7	58	[24]
Narrow	Φ7B	X. axonopodis pv. allii	12	7	58	[24]
Narrow	Φ14	X. axonopodis pv. allii	12	6	50	[24]
Broad	Φ16	X. axonopodis pv. allii	12	11	92	[24]
Broad	Φ17A	X. axonopodis pv. allii	12	11	92	[24]
Broad	Φ17B	X. axonopodis pv. allii	12	9	75	[24]
Broad	Φ31	X. axonopodis pv. allii	12	12	100	[24]
Polyvalent	Pg125	Xanthomonas strains	52	52	100	[69]
Polyvalent	Xcu-Pl	X. campestris pv. cucurbitae, X. campestris pv. dieffembachiae, X. campestris pv. holcicola	38	26	68	[72]
Polyvalent	Xcu-P3	X. campestris pv. cucurbitae, X. campestris pv. holcicola	38	17	45	[72]
Polyvalent	Xve-P1	X. campestris pv. pruni, X. campestris pv. vesicatoria	38	9	24	[72]
Polyvalent	Xca-P1	X. campestris pv. campestris, X. campestris pv. pruni	38	15	39	[72]
Polyvalent	Xhol-P1	X. campestris pv. cucurbitae, X. campestris pv. holcicola	38	15	39	[72]
Polyvalent	Xma-P1	X. campestris pv. cucurbitae, X. campestris pv. malvacearum	38	14	37	[72]
Polyvalent	Xpr-P1	X. campestris pv. holcicola, X. campestris pv. pruni	38	15	39	[72]

X Xanthomonas; pv pathovar

The polyvalent Xathomonas phage Pg125, is lytic to multiple strains from 25 species within the genus Xanthomonas [69]. Others in this category include phage Xcu-Pl, Xcu-P3, Xve-P1, and Xca-P1 which are lytic to Xanthomonas campestris pathovars (Table 3). The varied host ranges demonstrated by Xanthomonas phages imply that these lytic viruses can offer viable plant disease management alternatives. The high level of host specificity minimizes the risk of phage attack on beneficial bacteria [50].

Biology: physiological parameters

Incubation temperature, storage temperature, storage media

Incubation temperature

Xanthomonas phages can maintain their viability over a wide incubation temperature range. For example, Xanthomonas phaseoli phages (1, 20, 22, ΦPS, ΦSD, ΦSL, ΦRS, Φ56, Φ112, Pg60) remain viable between 2 and 28 °C [74]; Xanthomonas pruni phages (Xp3-A and Xp3-I) and Xanthomonas oryzae phages (Xp12 and φXOF4) between 20 and 50 °C [15, 23, 81] and Xanthomonas euvesicatoria phages (Kϕ1- Kϕ 15) between 35 and 70 °C [50].

Storage temperature

The storage temperature of Xanthomonas phages differs between strains. The initial titer 4 × 107 pfu/ml of phage Kϕ1, is maintained for 6 months when stored at + 4 °C in nutrient broth, compared to storage at + 20 °C where it declines to 2 × 107 pfu/ml within the same period [82]. Similarly, the lytic activity of Xanthomonas trifolii phages is maintained for a month at + 4 °C in phosphate buffer, pH 7 [73]. On the contrary, Xanthomonas arboricora phages (P6, P11, P15, P16, P20) survive poorly at + 4 °C in double distilled water during a one-year storage period. The initial phage titer (1 × 108 pfu/ml) drops drastically to 1 × 103 pfu/ml. The same phages decline to 8 × 104 pfu/ml when maintained at − 34 °C in the same media [52]. Therefore, Xanthomonas phages are maintained longer when stored at + 4 °C in nutrient broth. The appropriate storage conditions for different phages should be determined in order to ensure longevity of their effectiveness during storage and prior to biocontrol applications [83].

Storage media, ionic strength and pH

Phage viability is dependent on the storage media, ionic strength and pH and these have to be optimal to ensure phage longevity.

Different types of storage media have been investigated to understand their effects on phage viability. SM buffer is a mixture of sodium chloride (100 mM), magnesium sulphate (10 mM), tris-HCL (50 mM, pH 7.5) and gelatin (0.01%). In addition to SM buffer is nutrient broth, water/chloroform (H2O-CHCl3) and nutrient broth/chloroform (NB-CHCl3) combinations [52]. The initial phage titer (1 × 1010 pfu/ml) of Xanthomonas arboricora phages drops to 1 × 106 pfu/ml in SM buffer and to 1 × 105 pfu/ml in nutrient broth and water/chloroform during a one-year period at + 4 °C. In addition, phage titers decline further down to 1 × 104 pfu/ml under nutrient/chloroform combination [52]. In other studies, nutrient broth and SM buffer are favorable storage media for phage viability at + 4 °C for long-term storage. For example, the initial titer, 8 × 1010 pfu/ml of phage Kϕ1 declines slightly to 8 × 109 pfu/ml in nutrient broth and SM buffer at + 4 °C during a three-week storage period [82]. Further decline in phage titer of 3 × 109 pfu/ml is detected in sterile tap water and 10 mM magnesium sulphate while in distilled water the titers sharply fall to 3 × 107 pfu/ml at the same storage temperature and period [82]. Therefore, SM buffer is a better medium for phage survival than nutrient broth, tap water, magnesium sulphate, water/chloroform and nutrient broth/chloroform combinations [52]. The right storage media type will preserve the structural integrity of the phage and retain their infectivity during long-term storage [83].

The effect of ionic strength (salt concentration in liquid media) and pH on phage viability has been studied for a few Xanthomonas phages. Xp12 and Cf, lytic activity is maintained in distilled water or 0.1 M phosphate buffer, pH 7.0. However, the ability of these phages to lyse bacterial cells is prevented when they are stored in normal saline (0.9% sodium chloride) or 0.1 M citrate phosphate buffer, pH 7.0 [67, 84]. The optimal pH of Xanthomonas phages is between 5 and 11, with a number of phages being stable in acidic conditions such as pH 4 [23, 67, 82, 85].

Ultraviolet irradiation and chloroform resistance

The phyllosphere is a hostile environment and many factors such as ultraviolet (UV) irradiation prevent phage persistence and survivability [86]. As with all phages, Xanthomonas phages are inactivated by UV light. Formulations that increase phage survival consist of milk, corn and sucrose, minimizing UV-induced damages that result from the production of thymine dimers [82, 87, 88].

Chloroform treatment during isolation and enrichment process is used to release phage and kill host bacteria [89]. With the exception of Xf and Cf, many Xanthomonas phages are resistant to chloroform treatment because they lack a lipid envelope that surrounds the capsid. The organic solvent disrupts lipid membranes and inactivates the phage [23, 50, 52, 74, 82, 90]. The ability to resist chloroform denaturation makes non-enveloped Xanthomonas phages easy to isolate, culture and maintained for long-term storage [88].

Biology: life cycle, replication parameters and molecular mechanisms

Life cycle

Generally, clear plaques on a bacterial lawn could suggest that phages may have lytic life cycles, while turbid plaques represent temperate life cycles [91]. Xanthomonas phages produce both lytic and turbid plaques (Table 4). The latter outcome is due to the absence of bacterial host lysis resulting from phage genome integration into host bacteria chromosomes, causing latent infection [27]. Genome integration is facilitated by host XerC/D recombinases that mediate site-specific recombination of the phage genome into a 15 base-pair dif locus of the bacterial genome [93, 98]. Unlike lytic phages, temperate phages are not suitable for use as biocontrol agents due to their ability to cause lysogenic conversion, induction of superinfection immunity and increased risk of horizontal gene transfer [83].

Table 4

Life cycle of Xanthomonas phages

Phage	Life cycle	Host bacteria	Reference
Cp1	Lytic	X. axonopodis pv. citri	[92]
Cp2	Lytic	X. axonopodis pv. citri	[92]
XP3-A	Lytic	X. pruni	[15]
XP3-I	Lytic	X. pruni	[15]
Kϕ1	Lytic	X. euvesicatoria	[50]
Kϕ8	Lytic	X. euvesicatoria	[50]
Kϕ15	Lytic	X. euvesicatoria	[50]
Kϕ1–9 and Kϕ15	Lytic	X. euvesicatoria	[50]
Xoo-sp2	Lytic	X. oryzae pv. oryzae	[30]
Xoo-sp1–15	Lytic	X. oryzae pv. oryzae	[30]
Xp12	Lytic	X. oryzae pv. oryzae	[81]
X1	Lytic	X. oryzae pv. oryzae	[31]
X2	Lytic	X. oryzae pv. oryzae	[31]
X3	Lytic	X. oryzae pv. oryzae	[31]
X4	Lytic	X. oryzae pv. oryzae	[31]
X5	Lytic	X. oryzae pv. oryzae	[31]
φXOF4,φXOF1,φXOF 2,φXOF3, φXOT1,φXOT2,φXOM1	Lytic	X. oryzae pv. oryzae	[23]
P4L, P4M, P6M, P6M1, P14M, P14M1, P18M, P23M1,P33M, P37L, P37M, P37M1, P41M, P43M, P45M, P47M, P50M, P53M, P54M, P57M, P58M, P60M, P61M, P62M, P66M, P68M, P70M, P71L, P72M, P8L, P27L, P30L, P59L, P73L	Lytic	X. oryzae pv. oryzae	[22]
XTP1	Lytic	X. campestris pv. campestris	[71]
XC2	Lytic	X. campestris pv. campestris	[47]
Xcc9SH3	Lytic	X. campestris pv. campestris	[40]
P125	Lytic	Xanthomonas sp.	[69]
Xcu-P1	Lytic/Temperate	X. campestris pv. cucurbitae	[72]
Xcu-P3	Lytic/Temperate	X. campestris pv. cucurbitae	[72]
XholP1	Lytic/Temperate	X. campestris pv. holcicola	[72]
XmaP1	Lytic/Temperate	X. campestris pv. malvacearum	[72]
XcaP1	Lytic/Temperate	X. campestris pv. campestris	[72]
XprP1	Lytic/Temperate	X. campestris pv. pruni	[72]
XveP1	Lytic/Temperate	X. campestris pv. vesicatoria	[72]
P1 - P26	Lytic	X. arboricola pv. juglandis	[74]
1, 20, 22, ΦPS, ΦSD, ΦSL, ΦRS, Φ56, Φ112, Pg60	Lytic	X. phaseoli	[74]
Cf16	Temperate	X. campestris pv. citri	[93]
Cf1t	Temperate	X. campestris pv. citri	[94]
Cf16v1	Temperate	X. campestris pv. citri	[90]
ϕLf	Temperate	X. campestris pv. campestris	[95]
Cf1c	Temperate	X. campestris pv. citri	[96]
XacF1	Temperate	X. axonopodis pv. citri	[20]
Xf109	Temperate	X. oryzae pv. oryzae	[97]
XaF13	Temperate	X. vesicatoria	[45]
Xf	Temperate/carrier state	X. oryzae pv. oryzae	[68]
Cf	Temperate/carrier state	X. citri	[84]
ϕL7	Lytic	X. campestris pv. campestris	[95]

X Xanthomonas; pv pathovar; sp species

During adsorption, Xanthomonas phages bind to different bacteria host cell surface receptors [99]. The adsorption of phage ΦL7 onto Xanthomonas campestris pv. campestris requires binding to a complex receptor consisting of lipopolysaccharide and a secondary protein on the outer membrane.

Other filamentous phages such as Cf use the host pili (pilR) to bind to Xanthomonas campestris pv. citri [94, 100]. The phage then penetrates using chaperon proteins such as, TonB, ExbB, and ExbD1 encoded by operon, tonB–exbB–exbD1–exbD2 [101, 102]. The host bacteria are lysed by peptidoglycan glycohydrolase, which is located in the phage tail [103].

Replication parameters

The replication of phages is studied using the one-step growth experiment which measures the latent period and burst size of a phage on a specific bacterium. These are essential parameters in the description of phage properties. The latent period is the period between initial phage adsorption to a host cell to lysis and release of progeny viruses [91]. Xanthomonas phages have short latent periods ranging from 20 to 45 min to moderate periods, 60 to 90 min (Table 5). Very long latent periods ranging from 120 to 210 min occur for P125, Xoo-sp2, Xp12 (Siphorividae) and XTP (Myoviridae) (Table 5). The burst sizes range from 4.6 to 350 virions per infected cell (pfu/cell), with P125 showing the lowest burst size (4.6 pfu/cell) and Xoo-sp2 with the highest burst size (350 pfu/cell) (Table 5).

Table 5

Replication parameters of studied Xanthomonas phages

Phage	Host Bacterium	Family	Latent Period (Min)	Burst size (pfu/cell)	MOI	Phage Adsorption Temperature Time (min)	Reference
Cp1	X. axonopodis pv. citri	Siphoviridae	60	20	1	28 °C 10	[92]
Cp2	X. axonopodis pv. citri	Podoviridae	90	100	1	28 °C 10	[92]
P5	X. axonopodis pv. citri	n/a	40	60%	n/a	25 °C 20	[83]
Xp3-A	X. pruni	n/a	30–45	42–49	0.1	27 °C 20	[15]
Xp3-I	X. pruni	n/a	60–75	176–256	0.1	27 °C 20	[15]
Kϕ1	X. euvesicatoria	Myoviridae	20	75+/−4	0.1	27 °C 5	[50]
Kϕ8	X. euvesicatoria	Myoviridae	30	74+/−22	0.1	27 °C 5	[50]
Kϕ15	X. euvesicatoria	Myoviridae	30	70+/−11	0.1	27 °C 5	[50]
Xoo-sp2	X. oryzae pv. oryzae	Siphoviridae	180	350	0.1	28 °C 10	[30]
Xp12	X. oryzae pv. oryzae	Siphoviridae	140	35	0.1	28 °C -	[81]
X1	X. oryzae pv. oryzae	Myoviridae	20	88	10	30 °C 15	[31]
X2	X. oryzae pv. oryzae	Myoviridae	20	88	0.001	30 °C 15	[31]
X3	X. oryzae pv. oryzae	Myoviridae	40	50	0.01	30 °C 15	[31]
X4	X. oryzae pv. oryzae	Myoviridae	20	75	1	30 °C 15	[31]
X5	X. oryzae pv. oryzae	Myoviridae	20	100	1	30 °C 15	[31]
φXOF4	X. oryzae pv. oryzae	Siphoviridae	20–30	1.8 × 107 pfu/ml	0.1	28 °C 10	[23]
XTP1	X. campestris pv. campestris	Myoviridae	120	30–35	1	30 °C 15	[71]
X. phaseoli phage	X. phaseoli	Siphoviridae	30–45	40	n/a	22 °C 25	[104]
P125	Xanthomonas sp.	Siphoviridae	210	4.6	n/a	27 °C 30	[69]

X, Xanthomonas; sp., species; (n/a) not available in literature; min, minutes; MOI, multiplicity of infection; pfu, plaque forming unit; %, percentage; ml, milimeters

The multiplicity of infection (MOI) of reported Xanthomonas phages lie between 0.001 to 1, with the lowest observed for phage X2 at 0.001, and highest for X4, X5 and XTP1 at 1 (Table 5). It has been reported that phages with short latent period and high burst size have more efficient replication cycles [105]. Also, the optimal temperature and incubation time are essential parameters during phage adsorption. These conditions range between 22 and 30 °C, while incubation times are between 5 and 30 min for Xanthomonas phages (Table 5).

Molecular mechanisms

Phage-bacterial infection induces molecular changes that include DNA methylation, phosphorylation and transcription. DNA methylation is well-studied in phage Xp12 [81]. Upon infection in Xanthomonas oryzae pv. oryzae, Xp12 induces biosynthesis of an unusual base, 5-methylcytosine, that replaces all cytosine residues in the DNA of Xp12 [81]. The rest of the bases; adenine, thymine, and guanine, remain unaltered [67, 81]. DNA methylation confers unique physical and chemical properties upon Xp12 DNA i.e., acquisition of a low buoyant density and high melting temperature, compared to typical DNA [106]. The Xp12 phage-infected bacterial cells produce an enzyme deoxycytidylate methyltransferase, that catalyzes the direct methylation of deoxycytidine monophosphate (dCMP) to 5-methylcytosine, in the presence of tetrahydrofolic acid [107, 108].

Modification of phosphorylation occurs during Xanthomonas phage infection. When Xp12 infects Xanthomonas oryzae pv. oryzae, phosphorylation of three proteins is induced. The phosphorylated proteins 28 kDa, 28.5 kDa and 45 kDa in size are present only on infected cells. This type of molecular modification is suggestive of the existence of a phage specific regulatory mechanism involved during phage infection [109].

Transcriptional modifications are initiated upon phage-bacterial infection. In phage Xp10, infecting Xanthomonas oryzae pv. oryzae displays complete loss of transcription activity due deactivation of host RNA polymerase resulting from dissociation of the δ subunit from the host core RNA polymerase [110]. Later studies show that Xp10 reverts the transcription process by encoding an anti-termination factor p7 that allows formation of RNA transcripts by host RNA polymerase [111].

Biocontrol applications of Xanthomonas phages

This section explores several approaches where Xanthomonas phages are employed as biocontrol agents to manage Xanthomonas species in either greenhouse or field conditions. These methods have been successful at either inhibiting Xanthomonas growth or reducing disease severity. These include, but are not limited to use of monophages or cocktail treatments, phage mixtures with non-pathogenic or with pathogenic bacteria, phage combinations with antibiotics or plant inducers, UV- protectants and phage mutants [16, 21, 24, 30, 88, 112, 113].

To date, two Xanthomonas phage-based products are commercially available for the biocontrol of tomato, pepper spot and citrus canker [25]. The earliest evidence of Xanthomonas phage application was published in the early nineteenth century by Mallmann & Hemstreet [13], who determined that filtrate from decomposing cabbage applied to rotting cabbage inhibits the growth of Xanthomonas campestris pv. campestris in infected tissue. Since then, other forms of phage mixtures have been investigated.

Civerolo [114] applied crude lysates of lytic phage cocktail (Xp3-A and Xp3-I) on peach seedling foliage, 1–2 h before infection with Xanthomonas pruni under greenhouse conditions. Only 6–8% of leaves were infected, and the disease significantly reduced to 17–31% compared with 96% recorded on the water-treated control plants. In addition, application of either Xp3-A or Xp3-I mixed with Xanthomonas pruni and applied immediately before pathogen inoculation resulted in a 51–54% decrease of bacterial spot symptoms in peach seedlings under similar environmental settings. Therefore, the use of the phage cocktail significantly reduced disease severity better than single phage-pathogen mixture. This could be due to the synergy between the replication characteristics of both phages in the cocktail i.e. the latent period of Xp3-A and Xp3-I is 30–45 min and 60–75 min, whereas the burst size is 42–49 and 176–256 pfu/cell [114].

Some studies disagree with the evidence that supports the benefits provided by cocktail phage biocontrol of Xanthomonas associated diseases. In a recent study [24], spray application of a purified phage cocktail made up of three phages (ɸ16, ɸ17A, ɸ31) failed to inhibit the growth Xanthomonas axonopodis pv. allii, the causative agent of bacterial leaf blight of welsh onions. The cocktail treatment reduced infection of onion leaves to 43.3%, while a monophage phage treatment consisting ɸ31 reduced to 26.6% compared to the untreated, infected control leaves at 67.5% at 9 days after inoculation. Phage ɸ31, family Autographiviridae, had the broadest spectrum and lysed 12 out of 12 Xanthomonas axonopodis pv. allii strains, a trait that may contribute to its biological efficacy [24].

In another study [23], the phage φXOF4 inhibited the growth of Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight. The seedlings treated with φXOF4 at a titer of 1 × 108 pfu/ml showed no symptoms compared to 73% of the untreated group. Phage φXOF4, Siphoviridae, exhibited a broad host range where it lysed 6 out of 6 Xanthomonas oryzae pv. oryzae strains and had a short latent period between 20 and 30 min and a burst size that yields to the titer 1.8 × 107 pfu/ml. There is preference for cocktail phages because of their ability to effectively control pathogenic strains and delay the emergence of resistant strains [115, 116]; however, studies [23, 24] support the evidence that monophage treatment can be effective at disease reduction or elimination.

Applications of premixed phage-pathogen suspensions are further demonstrated by Dong [30], who observed low treatment outcomes in rice plants treated with Xoo-sp2 and Xanthomonas oryzae pv. oryzae suspension. The average lesion length in treated plants was 13.31 ± 1.69 cm compared to two control groups treated in sterile water (20.83 ± 2.43 cm) or skimmed milk (19.29 ± 2.07 cm). Phage Xoo-sp2 (Siphoviridae) had a broad host range where it lysed 9 out of 10 Xanthomonas oryzae pv. oryzae strains and had a latent period of 180 min and burst size of 350 pfu/cell. Although the authors considered only Xoo-sp2 out of the 15 phages, a phage cocktail should have been considered to improve biocontrol efficacy since the remaining phages displayed equally a broad host range where they lysed 9 out of 10 of the same strains.

Alternative control approaches using non-pathogenic bacteria and phage suspensions are demonstrated by Nagai [112]. The combination of non-pathogenic Xanthomonas strain (npX, AXCB1201) and phage (pXS, XcpSFC211) was sprayed on broccoli plants before inoculation of Xanthomonas campestris pv. campestris. The npX-pXS mixture significantly reduced disease severity to 18.9% compared with 86.2% by pXS alone and 93.7% of water-treated control plants in greenhouse settings. Field trials showed a decrease in disease severity albeit lower than the results from the greenhouse experiments. The npX-pXS mixture reduced the symptoms by 74% compared to 98% of water treated control plants or 86% of copper treated plants [112].

Integration of Xanthomonas phages with antimicrobials or UV-protectants has been explored as a disease management option. Borah [117] found that the combination of phage (XMP-1) and antibiotic (streptomycin) suppressed leaf spot of mungbean caused by Xanthomonas axonopodis pv. vignaeradiatae to 4% compared with 68% of the untreated seedlings. Moreover, seed germination increased to 86% in comparison to 75% in the untreated group. Furthermore, Balogh [88] applied formulated phages on tomato plants infected with bacterial spot incited by Xanthomonas campestris pv. vesicatoria. The phages were mixed with either 0.5% pregelatinized cornflour (PCF), casecrete NH-400 with 0.25% PCF, or 0.75% powdered skim milk with 0.5% sucrose. Phage treatment improved plant yield by 62% (skim milk), 51% (Casecrete), and 30% (PCF) compared to unformulated phages at 1% in greenhouse experiments. Under field experiments, phage treatment increased plant yield by 18% (skim milk), 32% (casecrete) and 23% (PCF) compared to unformulated phages at 14%. Therefore, skim milk gave better results in greenhouse experiments while casecrete performed better in the field. Similarly, Tewfike and Shimaa [66] found that formulated phages in skim milk controlled better bacterial halo blight symptoms of pepper caused by Xanthomonas axonopodis than with corn flour by 20.5 and 18.3% in the greenhouse and 19.5 and 32.2% in field conditions.

Some studies have shown that unformulated phages can control better plant diseases. Balogh [19] applied unformulated phages to citrus leaves infected with asiatic citrus canker and recorded an average of 59% reduction in disease severity in five greenhouse experiments. The same phage mixture in skim milk was not effective at controlling disease under similar environmental settings. In nursery experiments, unformulated phage treatment also reduced disease, but was less effective than copper-mancozeb, a chemical bactericide. Moreover, mixing the unformulated phages with copper-mancozeb achieved comparable results to unformulated phages alone [19]. Therefore different field settings (greenhouse, open field and nursery beds) should be considered during biocontrol studies because there is a possibility that phage efficacy depends on the field settings.

Plant inducers successfully control plant diseases, and therefore form an integral part of disease management practices. The application of mixtures of phages in skim milk/sucrose with Acibenzolar-S-methyl (ASM), a plant inducer, decrease the bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria under field conditions. The fruit yield of the formulated phage/ASM mixture was 67.9% compared to 60.8% of untreated control when applied twice biweekly in the first year [113]. Equally, Ibrahim [21] applied mixtures containing ASM and phages in skim milk/sucrose on citrus leaves for 4 days triweekly before inoculation of Xanthomonas citri subsp. citri, causative agent of asiatic citrus canker. Disease severity was reduced to 18.3% compared to 75.2% of the untreated control under greenhouse conditions. This observation agrees with results from field experiments where ASM/phages in skim milk/sucrose reduced disease to 12.5%, compared to 70.2% of the untreated control. When ASM was applied alone in the soil by drenching method, the disease was reduced to 38.2%, compared to 74.3% of the water-treated group after spraying 7 times triweekly before pathogen inoculation.

Mutated phages in formulations provide modest protection against plant disease compared with unformulated phages. The h-mutant phage mixtures (PMh; P4L, P43M, P23M1) in skim milk reduced bacterial blight disease of rice incited by Xanthomonas oryzae pv. oryzae to 18.1%, and wild type phage mixtures (PM; P4L, P43M, and P23M1) in the same formulation reduced the disease to 19.2%, compared to 39.1% of the untreated group. The mixtures were sprayed three times within an interval of 10 days. These tailed phages belong to the family Myoviridae and possess broad host range properties. Phage P4L lysed 33 out of 47, while P43M and P23M1 lysed 47 out of 47 Xanthomonas oryzae pv. oryzae strains [22]. Treatment with tecloftalam wettable powder, an agrochemical, demonstrated better results, with the disease symptoms reduced to 5% [22]. Therefore integration of tecloftalam wettable powder in plant protection could be a promising strategy for managing bacterial blight disease. On the contrary, agrochemicals have proved to be less effective than phages in controlling plant diseases. In a two-year greenhouse experiment, formulated phage DB1 in skim milk demonstrated improved black rot control by 71.1% while copper-based pesticide by 59.1%. Thus black rot caused by Xanthomonas campestris pv. campestris on cabbage seedlings can be successfully controlled by phage application [49].

Unformulated mutants reduce disease severity in infected plants. Flaherty [16] applied a mixture of host range mutant phages on tomato seedlings infected with Xanthomonas campestris pv. vesicatoria and symptoms of bacterial spot of tomato reduced to 0.9% compared to 40.5% of the untreated in the greenhouse. It increased the total weight of extra-large fruit by 14.9 and 24.2% in 1997 and 1998, respectively. Similarly, the severity of geranium bacterial blight declined when unformulated phage mutant mixtures were applied daily by foliar sprays on potted and seedling geraniums in greenhouse conditions [17].

Biofilm degradation is essential for the control of bacterial pathogenicity. The phage X3 causes 53% degradation of exopolysaccharide production and 43% biofilm degradation caused by Xanthomonas oryzae pv. oryzae that causes bacterial blight of rice [31]. When phage X3 was sprayed on rice plant foliage and seeds before pathogen inoculation, the plants improved by 83.1 and 95.4%. The phage X3 did not perform well when applied after pathogen inoculation, with results recorded between 28.9 and 73.9% [31]. Phage X3, family Myoviridae, had the broadest host range, lysed 22 out of the 23 Xanthomonas oryzae pv. oryzae strains tested and had the most extended latent period of 40 min with a burst size of 50 pfu/cell [31]. Likewise, infection of XacF1 (Inoviridae), a temperate phage, pathogenic to Xanthomonas axonopodis pv. citri, causing asiatic citrus canker, inhibits xanthan production, a component of extracellular polysaccharide that exacerbates the disease. The lesions on leaves sprayed with XacF1 reduced to 1 mm in width compared to 6.5 mm in untreated leaves. Therefore, the reduction in xanthan production caused by XacF1 phage reduces disease symptoms [20].

The frequency of phage spray and contact time on plant surfaces are factors investigated to improve the efficacy of phage applications. Lang [18] showed that multiple applications, i.e. biweekly or weekly applications of phages, effectively reduce symptoms of leaf blight of onion caused by Xanthomonas axonopodis pv. allii to 50%. Similar results were obtained when copper hydroxide-mancozeb was sprayed weekly on onion plants. Furthermore, biweekly application of Acibenzolar-S-methyl and phages reduced the disease by up to 50%. Hence, biweekly spray schedules are a promising strategy for sustainable control of leaf blight of onion.

Successful control of plant diseases is directly linked to the contact time of phages on plant surfaces. Gašić [82] successfully controlled bacterial pepper spot caused by Xanthomonas euvesicatoria by allowing a long contact time of phage Kϕ1 (Myoviridae) on plant leaves. The longest time of phage contact was 2 h before and 15 min after pathogen inoculation. This resulted in an average lesion number of 157, 213, and 189 compared to 332, 422, and 567 of the untreated control in three greenhouse experiments. The contact time experiments were further tested on copper hydroxide mixed with Kϕ1. At a contact time of 26 h before pathogen inoculation, a significant reduction in average lesion number was observed with scores of 63, 41, and 66 compared to 332, 422, and 567 of the untreated control. Thus longer contact time of phage Kϕ1 on plant surfaces allows effective control of pepper bacterial spot. There is a direct relationship between the timing of phage application and the efficacy of disease control. Evening applications of phage on foliage achieve better disease control since this period minimizes phage exposure to UV irradiation and extends phage longevity [88]. Phage Kϕ1 had the broadest host range where it lysed 59 out of 59 Xanthomonas euvesicatoria strains [50] and had a latent period and burst size of 20 min and 75 phage particles per infected cell respectively. Its multiplication and broad lytic abilities may contribute to its success at managing pepper bacterial spot.

The study of phage lysins as alternative biocontrol for Xanthomonas phytopathogens is rarely reported. One study has shown that phage lysozyme, Lys411, encoded by the genome of Xanthomonas oryzae phage, ϕXo411, can lyse Xanthomonas strains, making the protein a candidate with potential to control plant diseases caused by Xanthomonas [118].

One of the limitations faced by plant-based phage application is the hostile environment of the phyllosphere, where phages degrade rapidly due to desiccation or UV light. Phage formulations demonstrate protective benefits that enhance phage longevity and antibacterial activity [19, 88]; however, not all phages are effective in UV protectants [19]. Although, leaf surfaces of some plants do support phage multiplications, others do not; and this could potentially have adverse effects on the efficacy of a biocontrol product. Balogh [119] found that two Xanthomonas perforans phages (ɸXv3–21 and ɸXp06–02) multiplied and maintained populations on tomato leaf surface but did not achieve the same level of multiplication on grapefruit leaves. More research is needed to understand plant compounds involved and the mechanisms involved in this plant-phage interaction.

Conclusion

Several Xanthomonas phages are evaluated for their potential as biocontrol agents against Xanthomonas species. So far, most of these belong to order Caudovirales and are lytic to a broad range of host strains. They are isolated from diverse ecosystems and distributed across the globe depending on the presence of the pathogen they infect. Their structural integrity and functionality in in vitro conditions is maintained under optimal growth and storage conditions. Pathogenesis of Xanthomonas phages in bacteria induce molecular alterations that may have regulatory functions important during their life cycle. Although few studies have focused on this aspect of biology, more research is needed to understand their life cycle.

From their first discovery in filtrates to applications as phage/pathogen suspensions, or in combination with other antimicrobials or with UV-protectants or as cocktail/monophage treatments, phages have proved to be promising alternatives to agrochemicals and antibiotics. They can reduce disease severity or inhibit bacteria growth in diverse field settings. So far, two Xanthomonas phage-based biocontrol products are commercially available for plant disease control. As the transition into commercial products continues, more studies are needed to tap into the many unexploited potentials of Xanthomonas phages for a range of Xanthomonas related plant diseases.

Additional file 1 : Table S1 Taxonomic classification, genomic properties and host bacteria of Xanthomonas phages. Description of data: Xanthomonas phages of order Caudovirales and Tubulavirales, their morphological and genomic properties and host bacteria.