Abstract

Introduction

Pomegranate (Punica granatum L.) is an ancient fruit crop, has gained worldwide popularity due to its high nutraceutical and medicinal value. Pomegranates are rich in antioxidants, antibacterial, antifungal properties, and hence touted as a “superfood” (Johanningsmeier and Harris 2011). Cultivation of pomegranate is attracting more farmers due to its hardy nature, wide acclimatization to varied soil and climatic condition, and tolerance to salt and drought stress. Consistent demand in the national and international markets also widened the scope for earning higher remunerative value by pomegranate cultivation. In recent years, total crop production is severely compromised due to bacterial blight (BB) infection caused by Xanthomonas axonopodis pv. punicae (Xap) (Sharma et al. 2015). Benagi and Kumar (2009) recorded the high yield loss from 1.8 lakh tonnes to less than 10,000 tonnes per annum in 2007–2008 accounting for revenue loss of about Rs. 200 crores in India. BB disease was a minor threat in the beginning when it was first reported, but now it has become an epidemic infecting all the major areas of pomegranate cultivation and hampering production in India (Sharma et al. 2015). The disease is also reported in Turkey, Pakistan, and South Africa (Icoz et al. 2014; Akhtar and Bhatti 1992; Petersen et al. 2010) and slowly spreading across the world, making an international issue of pomegranate (Fig. S1).

The infection of BB can be seen on leaves, twigs, and fruits. Typical symptoms include oily water-soaked lesions on leaves and fruit, later the same developing into brown-to-black necrotic lesions with irregular cracks (Singh et al. 2015). The entry of the pathogen generally occurs through natural openings like stomata, hydathodes, lenticels, and wounds. Under field conditions the pathogen spreads mechanically through rainwater splashes, insects, vectors, improper water irrigation systems, and poor management practice (Sharma et al. 2015). Xap is a versatile pathogen, can survive in plant debris, soil with minimum moisture for a long time (Sharma et al. 2015) resulting in an easy spread of the pathogen. Most plant pathogens can infect a wide range of plant species and that referred to as alternate hosts which act as a reservoir for pathogen and increase the pathogen spread (Farr et al. 2004). Developing an effective disease management strategy is very necessary to understand pathogen biology and its wide host range. Biochemical and molecular-based characterization has been widely used for the identification of the pathogen and effector-based avirulence (Avr) genes provide the most accurate tool for molecular confirmation of the pathogen (Doddaraju et al. 2019; Block et al. 2008).

Domestication and modernization of agricultural practices have drastically reduced the genetic variation of the crop by replacing it with high yielding and these high-yielding varieties are susceptible to various pests and diseases (Tanksley and McCouch 1997; Samal and Rout 2018). In pomegranate, Bhagwa, Arakta, Ganesh, and Mridula are the most popular cultivars having maximum yield and high commercial value but, none of them are resistant/tolerant to bacterial blight (Priya et al. 2016). Hence, identification of novel resistance sources and utilizing them in breeding of resistant cultivars, is on high priority. Under the limitations of poor resistance sources, several integrated disease management (IDM) protocols have been demonstrated for the effective management of BB in pomegranate (Anonymous 2008; Benagi and Kumar 2009). These IDM protocols include a combination of chemical antibiotics/pesticides that are harmful to the environment, and the application of these chemicals requires a huge per-capita investment.

Plants have evolved with both acquired and innate immunity to combat various biotic stresses. Interactions between plant and pathogen develop reactive oxidative burst, leading to cell death. During pathogen invasion, local responses in plants include the change in cell wall composition, lignin production, cell wall thickening, and synthesis of the antimicrobial component such as pathogen-related proteins (PR proteins) and phytoalexins (Slusarenko et al. 2000). These responses are triggered by several signaling compounds or elicitors molecules mediated through, salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and/or ethylene (ET) pathway during pathogen combat (Slusarenko et al. 2000; Wang et al. 2019; You et al. 2011; Kashyapa et al. 2018). Elicitor molecules induce systemic resistance in various crops, such as glycoproteins, oligosaccharides, lipids, X-glucans, and chitin oligomers (Aziz et al. 2003). Due to their potential role as plant protectants, these elicitor molecules have been the most effective tools for systemic disease control in various crops (Hayat et al. 2012; Justyna and Ewa 2013; Xin et al. 2019; Kashyapa et al. 2018).

With all these prospects in the present study, 149 rare pomegranate germplasm have been collected from different geographical locations. They were screened against Xap and evaluated the expression level of pathogen-triggered immune responses against BB. Further, in search of a novel alternative strategy for disease management, different elicitor molecules that are known to induce systemic resistance in the various plant species were evaluated under the greenhouse condition to know their efficacy in controlling BB.

Materials and methods

Results

Isolation and pathogenicity of the pathogen

The pathogen was successfully isolated from infected tissue samples. A typical colony of yellow, circular, convex, mucoid, pin-headed appear after 48 h of incubation were selected and further sub-cultured on NG medium (Fig. S2B). Pathogenicity of the isolated culture was proved as per Koch's postulates. Upon artificial inoculation, plants exhibit typical characteristics symptoms of oily water-soaked lesions on leaves, gradually developed into yellow hallow, and turn to dark brown lesions, water-treated control plant did not show any symptom of pathogen infection (Fig. 1).

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Fig. 1

Pathogenicity study of isolated bacterial culture Xanthomonas axonopodis pv. punicae in pomegranate plant (cv. Bhagwa) under greenhouse condition. a Control plant inoculated with water. b Infected plant showing typical symptoms of bacterial blight on 9th day post-inoculation. c Test plant showing typical symptoms of bacterial blight on 20th days post-inoculation

Molecular identification of Xap

The molecular identity of the pathogen was established using XopQ effector protein primer. Exact amplification at 190 bp of the partial gene sequence in gDNA of Xap confirmed the isolate as Xap (Fig. 2). The sequence details were submitted to NCBI gene bank and the Accession number is availed KX702398.1.

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Fig. 2

PCR amplification of XopQ effector-based primers from gDNA isolated from a pure culture of Xanthomonas axonopodis pv. punicae. Lane, M=GenRuler mix 1 kb, 1=XopQ primer amplification at 190 bp

Evaluation of pomegranate germplasm

A total of 149 pomegranate germplasm were screened for BB under the greenhouse condition by artificial inoculation technique. All the screened lines recorded the disease to the BB pathogen Xap, with varying susceptibility levels. Among the screened lines, a large majority of lines recorded high susceptibility recording disease severity of more than 25%, and commercially cultivated Bhagwa recorded the highest disease severity of 58.11% followed by Bedanasedana, Mridual, and Ruby with 55.81, 54.43, and 49.87% of disease severity, respectively (Table ​(Table3).3). Among 149 genotypes, 31 genotypes are moderately susceptible to the pathogen and these include most of the EC series and few IC series genotypes. Seven genotypic lines recorded the least severity and were categorized as tolerant lines, where IC318735 recorded the lowest disease severity with 4.91% followed by IC318724 and IC318762 and with 5.66 and 6.82% of disease severity, respectively. Frequency distribution analysis also indicated a varied level of disease severity in screened lines with mean disease severity of 30.67% and a standard deviation of 9.22 (Fig. 3).

Table 3

Summary of pomegranate germplasm/accessions response to bacterial blight pathogen under greenhouse

Sl. no.	Accession no.	Disease severity (%)	Phenotypic disease observation			Disease reaction
			Day6	Day9	Day20
1.	Bhagwa	58.11	√	√	√	Highly susceptible
2.	Bedanasedana	55.81	√	√	√
3.	Mridula	54.43	√	√	√
4.	Ruby	49.87	√	√	√
5.	Bosekaliniski	46.82	√	√	√
6.	Arakta	44.49	√	√	√
7.	Sural Anar	43.12	√	√	√
8.	Ganesh	43.17	√	√	√
9.	K.R. S	42.59	√	√	√
10.	P-26	42.62	×	√	√
11.	Sural Anar	42.42	×	√	√
12.	Cranado-de-Etcho	42.23	√	√	√
13.	Sursakkar	42.14	×	√	√
14.	Shirin Anar	41.48	√	√	√
15.	Coimbatore White	41.25	×	√	√
16.	Speensakarin	40.69	×	√	√
17.	Kabul	40.55	√	√	√
18.	Masta	40.51	√	√	√
19.	Kazki Anar	40.49	√	√	√
20.	Kabul yellow	40.25	×	√	√
21.	Gul-e-shah rose pink	40.08	×	√	√
22.	Kabul	39.71	√	√	√
23.	IC318787	39.57	×	√	√
24.	Bedan thin skin	39.38	√	√	√
25.	Gal-e-shah red	39.31	√	√	√
26.	Jodhpur red	39.27	×	√	√
27.	Jyoti	38.89	×	√	√
28.	Achik Dana	38.80	√	√	√
29.	Dholka	38.59	√	√	√
30.	A.K.Anar	38.32	×	√	√
31.	P-23	38.40	×	√	√
32.	G-137	38.30	×	√	√
33.	Kaladagi Local Tidagundi	38.21	×	√	√
34.	EC798766	38.01	×	√	√
35.	Kabul Conoor	37.94	√	√	√
36.	IC318766	37.54	√	√	√
37.	EC798759	36.61	√	√	√
38.	EC798751	36.57	√	√	√
39.	Yercaud	36.19	√	√	√
40.	Kabul	36.02	√	√	√
41.	Kaladagi local	36.18	√	√	√
42.	RCR CB Plot	35.80	×	√	√
43.	Yercaud HRS	35.63	√	√	√
44.	IC318705	35.40	√	√	√
45.	P-13	35.15	√	√	√
46.	Baiseen seedless	35.16	×	√	√
47.	EC798793	35.15	×	√	√
48.	EC798740	35.31	×	√	√
49.	IC318784	34.66	√	√	√
50.	Lupinia	34.49	√	√	√
51.	Alandi	34.43	√	√	√
52.	IC318797	34.29	√	√	√
53.	P-16	34.22	×	√	√
54.	IC318791	34.11	×	√	√
55.	Kazakali Anar	33.56	×	√	√
56.	EC798851	33.46	×	√	√
57.	Patna- 5	33.17	×	√	√
58.	Soft grafted	33.14	×	√	√
59.	EC798731	32.91	×	√	√
60.	IC318775	32.52	×	√	√
61.	IC318717	32.50	×	√	√
62.	IC318736	31.84	×	√	√
63.	IC318752	31.82	×	√	√
64.	EC798783	30.73	×	√	√
65.	EC798798	31.72	×	√	√
66.	Domani	31.45	×	√	√
67.	Alah	31.24	×	√	√
68.	Tabesto	31.21	×	√	√
69.	EC798758	31.20	×	√	√
70.	Speen Danedar	31.01	×	√	√
71.	Bedana Suri (B)	31.29	×	√	√
72.	EC798807	31.20	×	√	√
73.	EC798789	31.11	×	√	√
74.	IC318708	31.12	×	√	√
75.	EC798797	31.32	×	√	√
76.	IC318697	30.42	×	√	√
77.	EC798850	30.14	×	√	√
78.	Kanamadi 2	30.52	×	√	√
79.	Dorasata Malas	30.09	×	√	√
80.	EC798801	30.46	×	√	√
81.	EC798749	30.06	×	√	√
82.	Yercaud Local	29.96	×	√	√
83.	Gul-e-shah	29.63	×	√	√
84.	Bedana Suri (A)	29.61	×	√	√
85.	Agah	29.19	×	√	√
86.	EC798754	29.79	×	√	√
87.	EC798762	29.69	×	√	√
88.	EC798829	29.80	×	√	√
89.	IC318701	29.15	×	√	√
90.	EC798843	29.76	×	√	√
91.	Jalore seedless	28.81	×	√	√
92.	Kali Shirin	28.75	×	√	√
93.	EC798821	28.51	×	√	√
94.	EC798833	28.32	×	√	√
95.	EC798840	28.44	×	√	√
96.	EC798730	28.11	×	√	√
97.	EC798838	28.34	×	√	√
98.	IC318758	28.21	×	√	√
99.	EC798828	28.46	×	√	√
100.	IC318733	28.61	×	√	√
101.	Sihaishirin	27.39	×	√	√
102.	EC798836	27.92	×	√	√
103.	EC798776	27.45	×	√	√
104.	IC318776	27.98	×	√	√
105.	EC798773	27.93	×	√	√
106.	IC318738	26.39	×	√	√
107.	IC318738	26.39	×	√	√
108.	EC798729	26.39	×	√	√
109.	EC798723	26.19	×	√	√
110.	EC798767	26.14	×	√	√
111.	IC318760	25.64	×	√	√	Moderately susceptible
112.	EC798746	25.46	×	√	√
113.	IC318709	24.36	×	×	√
114.	IC318759	24.51	×	√	√
115.	EC798832	24.27	×	√	√
116.	IC318700	23.25	×	×	√
117.	EC798784	23.03	×	√	√
118.	EC798806	23.46	×	√	√
119.	EC798772	23.19	×	√	√
120.	EC798768	23.33	×	√	√
121.	IC318703	22.10	×	×	√
122.	EC798774	22.82	×	×	√
123.	EC798810	22.90	×	√	√
124.	EC798734	22.67	×	√	√
125.	EC798804	22.67	×	√	√
126.	EC798842	22.21	×	√	√
127.	EC798756	22.78	×	√	√
128.	IC318754	21.59	×	√	√
129.	IC318798	21.09	×	√	√
130.	IC318767	20.94	×	√	√
131.	EC798796	20.59	×	√	√
132.	EC798742	20.07	×	√	√
133.	EC798765	20.42	×	√	√
134.	IC318790	19.99	×	√	√
135.	EC798735	20.87	×	√	√
136.	EC79882	19.59	×	√	√
137.	EC798780	19.01	×	√	√
138.	IC318716	18.57	×	×	√
139.	IC318732	18.19	×	√	√
140.	IC318751	19.06	×	√	√
141.	IC318741	18.21	×	×	√
142.	IC318706	14.78	×	√	√
143.	IC318712	11.69	×	×	√	Tolerant
144.	IC318734	10.52	×	×	√
145.	IC318707	8.63	×	×	√
146.	ACC8	8.43	×	×	√
147.	IC318762	6.82	×	×	√
148.	IC318724	5.66	×	×	√
149.	IC318735	4.91	×	×	√

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Fig. 3

Frequency distribution of disease severity of pomegranate germplasm screened for bacterial blight disease under greenhouse condition

Transcript analysis of defense response upon pathogen attack

The host defense response to pathogen attack was determined by qPCR analysis of PAL, CS3, chitinase, PR1, and PR10 genes in tolerant lines IC318735 and IC318724. A total of 5 defense responsive genes known to express in different pathways were evaluated at 0, 24, 72, and 216-h post-inoculation (hpi), and data were evaluated in comparison to the most susceptible variety Bhagwa. Upregulations of CS3, chitinase, and PR10 were found to be highly expressed between 24 to 72 hpi, whereas high expression of PAL and PR1 were recorded at 216 hpi. Upregulation of PR1 in IC318735 recorded high at 216 h with 59.71-fold followed by 42.5- and 5.1-fold at 24 and 72 hpi, respectively. However, in IC318724 maximum upregulation of the PR1 gene was observed at 24 hpi (23.5-fold) and 216 hpi (19.16-fold), respectively. In expression analysis of PAL gene, an interesting pattern was observed in both the lines, IC318735 recorded high at 24 hpi (13.83-fold), and gradually decreases at 72 and 216 hpi with 2.4- and 3.9-fold, whereas in IC318735, initial upregulation was observed at 24 hpi (2.2-fold), further elevated at 72 hpi (6.68-fold) and 216 hpi (6.32-fold), respectively. In contrast, Callose synthase was recorded high at 72 hpi in both the lines IC318735 (9.8-fold) and IC318724 (8.7-fold), followed by twofold at 216 hpi and 1.16-fold in IC318735 and no significant difference in other time points of IC318724. Chitinase gene in IC318735 recorded maximum upregulation at 24 hpi with 13.7-fold, followed by 2.15- and 2.46-fold at 72 and 216 hpi. A similar pattern was observed in IC318724, where expression of chitinase recorded high at 24 hpi with threefold followed by 1.8-fold at 72 hpi, respectively. The expression pattern of PR10 was recorded high first in IC318735 at 24 hpi with 4.26-fold followed by a gradual decrease at 72 and 216 h by 2.66 and 1.43-fold, respectively. However, in IC318724 upregulation of the PR10 gene was recorded only at 24 and 72 hpi with 1.5- and 2.5-fold change, respectively. The overall representation of the data is represented in Fig. 4.

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Fig. 4

Relative expression of defense responsive gene in IC318735 and IC318724 determined by qRT-PCR during pathogen attack. Total RNA was reverse transcribed into cDNA and used as a template for qRT-PCR as described in “Materials and methods”. The bar indicates the standard error

Discussion

Pomegranate is an important fruit crop of tropical and subtropical parts of the world, significantly contributes to the global economy (Mastrogiannidou et al. 2016). However, modernization of agricultural practice, adoption of monoculture reduced the genetic variability among the crops leading to increased pests and diseases. Biotic stresses like bacterial blight (Xanthomoas axonopodis pv. punicae), fungal wilt (Ceratocystics sp.), leaf spot (Cercospora punicae), anthracnose (Colletotrichum gloeosporioides), aspergillus fruit rot (Aspergillus niger), and heart rot (Alternaria alternate), are the most common threats for pomegranate cultivation. Among these, bacterial blight is the most serious limiting factor for pomegranate cultivation leading to 60–80% yield loss.

Molecular and biochemical identification of a pathogen is very necessary for working an effective disease management strategy in any crop including breeding of resistance. BB pathogen is sensitive to salt, pH, and temperature. The optimum temperature required for the growth of the pathogen is between 26 and 32 °C. Xap produces a unique brown fuscan pigmentation, that appears 10-day post-inoculation, similar observations were confirmed by the national research center of pomegranate (Anonymous 2008). Molecular confirmation of the pathogen using an effector-based primer specific to Xanthomonas spp. provides accurate identification of the pathogen, since Xanthomonas axonopodis pv. punicae is the only Xanthomonas species infecting pomegranate. Effector-based avirulence (Avr) genes are the unique conserved sequences that are the most appropriate candidate genes for the identification of plant pathogens (Block et al. 2008).

Array of plant pathogens are fast evolving, they can breakdown the plant resistance/immunity resulting in increased disease epidemics in multiple hosts. For instance, Xanthomonas citri spp. citri is mainly known to cause citrus canker in citrus lemon and reported to infect several host species such as lime, grapefruit, sweet orange, and many more of the citrus family (Ference et al. 2018). Likewise, pathogen Xanthomonas campestris pv. campestris causing black rot disease of cabbage also reported infecting plants belonging to all the crucifer families (Bhat et al. 2010). To understand the host specificity of Xap, ten different plant species were selected. Cotton, lemon, cabbage, tomato, betel, banana, and chilli were selected based on being host to other Xanthomonas spp. Neem and pongamia plants were selected based on earlier reports of being a host of Xap (Yenjerappa 2009). In contrast to the study of Yenjerappa (2009), no plants other than pomegranate infected with Xap, indicating Xap is specific to pomegranate. In vice versa, three closely related Xanthomonas spp. Viz. Xanthomonas citri spp. citri, Xanthomonas campestris pv. campestris and Xanthomonas campestris pv. malvacearum were cross-inoculated on healthy pomegranate plants, to check the alternate pathogen that may cause bacterial blight infection in pomegranate. Here also no Xanthomonas spp. other than Xap, able to produce BB symptoms in pomegranate plants, indicating BB of pomegranate is only caused by Xap. Host range of a pathogen studies offers several advantages such as understanding the life cycle of a pathogen, developing disease diagnostics tools, and elucidating defense response against the pathogen. This knowledge serves as a valuable asset to the plant breeding approach (Hollings 1959; Bebber 2015) and designing strategies for plant disease management. In pomegranate, several commercial cultivated lines are developed that are attributed to high yield and nutritional value, but none of them are resistant to bacterial blight. Current management practice includes the use of a large volume of synthetic antibiotics that require huge economic investment and had repercussions to surrounding environmental toxicity with mere protection against the pathogen. Further, the use of excessive synthetic pesticides leads to the development of resistance in the pathogen that may worsen the situation of disease management. Hence, developing a new variety via a resistant breeding program is the most viable option. However, this requires large-scale screening of local and wild variety for identification of suitable tolerant or resistance lines. In pomegranate, Priya et al. (2016) screened different pomegranate genotypes for resistance using detached leaf assay and reported IC318734 as a putative resistant line of pomegranate. In our study, pomegranate lines were screened under the greenhouse condition through spray inoculation of the pathogen. All 149 screened lines exhibited susceptibility to the pathogen. However, IC318735 and IC318724 recorded the lowest disease severity of 4.91 and 5.66%, respectively (Table ​(Table3).3). Variation in disease severity level may be due to the accumulation of various secondary metabolites and stomatal opening or closing during pathogen attack. Priya et al. (2016) stated that fewer stomatal pores are the major contributing factors for disease tolerance in putative lines of resistant pomegranate.

In plants, resistance, or tolerance towards the pathogen depends on the gene for gene interaction between the pathogen avirulence (Avr) and host resistance (R) gene (Flor 1971; Block et al. 2008). The absence of these genes or delayed expression of these may lead to disease tolerance or susceptibility to pathogen invasion. Thus, tolerance to the disease depends upon the activation of various defense cascades of genes during pathogen attack, and this mechanism is called systemic acquired resistance (SAR) (Slusarenko et al. 2000; Van Loon et al. 2006). In understanding the defense responses in tolerant lines of pomegranate, we observed the higher expression levels of PR1, PR10, PAL, CS3, and chitinase upon pathogen attack.

PR proteins are an important part of plant defense response highly expressed during pathogen attack, accumulation of PR proteins can be observed locally around the infected part of the plant and, also in remote healthy tissues (Van Loon et al. 2006). Accumulation of PR proteins in the healthy parts of the plant will suppress the further spread of the pathogen. In pomegranate inoculation with Xap triggered the expression of PR1, PR10, and chitinase to the maximum between 24 and 72 h in IC318735 and IC318724 lines compared to Bhagwa, indicating active involvement of these genes in disease tolerance. Fang et al. (2019), reported the over-expression of the PR1 gene as part of disease resistance in mulberry. Alexander et al. (1993) reported the active role of PR1 against oomycete pathogens in transgenic tobacco, similarly, Choi et al. (2012) stated overexpression of PR10 increased resistance against Hyaloperonospora arabidopsidis and P. syringae pv. tomato in Arabidopsis. Jwa et al. (2001) also report that overexpression of PR1 and PR10 in rice increased defense response against Magnaporthe grisea. Likewise, several research reports stated that over-expression of PR genes conferring disease resistance towards bacterial, fungal, and in some viral pathogens (Cutt et al. 1989; Dolatabadi et al. 2014; Liu et al. 2019).

PAL is the first step in the phenylpropanoid pathway leading to the production of lignin and many secondary metabolites such as phytoalexins, flavonoids, and coumarins against various biotic and abiotic stress. PAL is the active intermediate gene in the synthesis of salicylic acid, catalyzing the conversion of phenylalanine to trans-cinnamic acid (Apel and Hirt 2004). In the present study, inoculation of Xap in tolerant lines of pomegranate recorded the upregulation of PAL between 72 and 216 h indicating the active response during pathogenesis. Chithrashree et al. (2011) recorded the higher activity of PAL in Bacillus subtilis treated rice seedlings after pathogen inoculation, Farahani and Taghavi (2017), reported accumulation of PAL might have an active role in conferring disease resistance against Xanthomonas axonopodis pv. phaseoli in tomato. Chitinase is the most important hydrolytic enzyme that inhibits the activity of the pathogen by cell wall degradation (Ebram et al. 2011). In tolerant lines of pomegranate upregulation of chitinase was observed maximum at 24 h, in both the lines, and subsequently, no significant upregulation was recorded.

Strengthening the plant cell wall is a prime defense response for restricting the entry of the pathogen into the host system. During the pathogen attack, the deposition of callose occurs between the plasma membrane and cell wall at the site of pathogen infection (Nishimura 2003). In the present study, higher expression of callose synthase gene in both the tolerant lines of pomegranate when compared to control and maximum upregulation of CS3 gene was observed at 72 h in both tolerant lines of pomegranate, which is the key time for the pathogen entry into the host system. In support of the present study, Kumar and Mondal (2013) reported the deposition of callose which acts as a structural barrier in pomegranate against Xap. From the above data, it is very clear that systemic acquired resistance of host immunity directly contributing to the pathogen resistance in tolerant lines pomegranate.

Apart from the pathogens, many elicitor molecules also have been identified that trigger the immune response against pathogens in higher plants. Foliar application or soil amendment of these molecules resulted in the production of phytoalexin biosynthesis, production of reactive oxygen species, strengthening plant cell wall, callose deposition, defense enzyme synthesis, and production of PR proteins, that induce systemic resistance against the pathogen (Van Loon et al. 2006; Wang et al. 2019; Algam et al. 2013). The application of different elicitor molecules shows significant disease protection against Xap in pomegranate. Foliar application of Proline, GABA and, chitosan at 600 ppm shows maximum disease protection of 89.59, 88.59, and 87.15%, respectively, when compared to control. Proline is an amino acid precursor to a protein known to play a significant role in plant–pathogen interaction. Hayat et al. (2012) state that proline acts as an antioxidative defense molecule, metal chelator, and signaling molecule during pathogenesis. Similarly, the application of chitosan induces ISR in higher plants, like generating reactive oxygen species, strengthening cell wall by lignification, biosynthesis phytoalexin, and jasmonic acid signaling leading to disease tolerance (EI Hadrami et al. 2010). Followed by Proline, application of chitosan, non-protein amino acids GABA (gamma-aminobutyric Acid) and BABA (β-aminobutyric acid) also recorded a significant reduction in disease severity when compared to control. Inducer property of GABA and BABA against various phytopathogens has been well documented in Arabidopsis, potato, grapefruit, and lime against various phytopathogens (Justyna and Ewa 2013; Wang et al. 2019). Satková et al. (2016) reported that application of BABA induces systemic resistance against powdery mildew of tomato Oidium neolycopersici in tomato by activation of ethylene-dependent signaling mechanism. In a similar study, Kashyapa et al. (2018), demonstrated that foliar application BABA can induce systemic resistance against Tilletia indica in wheat. Cohen (2002) reports that the application β-aminobutyric acid lowers the disease by upregulating the expression level of PR proteins, thus inducing systemic resistance in plants. Wang et al. (2019) stated that the biosynthesis of GABA plays a vital role in plant–pathogen interactions between Ralstonia solanacearum and tomato plants. Likewise, another tested inducer molecule laminarin, eugenol, isonicotinic acid, paclobutrazol, and salicylic acid found effective against BB by recording the lowest disease severity as several reports suggest the positive effect of these inducers in plant disease management (Xin et al. 2019; You et al. 2011; Eid 2013; Wang et al. 2019). Our results indicate the all the tested inducer can be effectively used for the management of BB in pomegranate under controlled conditions. However, these metabolites need to be further tested for their efficacy under field conditions and incorporate into a suitable formulation to make the effective use of these elicitors in integrated disease management practices in pomegranate.

Conclusion

The study indicates that Xanthomonas axonopodis pv. punicae is highly specific to pomegranate, and there are no alternate hosts to this pathogen. Resistance to the pathogen in tolerant lines of pomegranate (IC318724 and IC318735), resulted from the expression of high transcripts level of defence response genes. Further, foliar application of GABA, BABA, chitosan, laminarin, and eugenol resulted in significant protection against the pathogen. These molecules can be effectively employed in the IDM module for disease management also the development of novel biological/SAR formulation.

Supplementary Information

Acknowledgements

Author contribution

PK designed the experiment, conducted pathogen isolation, challenge inoculation, varietal collection and screening, molecular experiments, and wrote the manuscript. MSD did biochemical analysis and pathogen identification. PD analyzed data and help to rewrite the manuscript. BSM supervised and guided for the experiment. MG designed and supervised the whole experiment and guided for conducting studies and edited the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

References