Nutrient imbalance indices are closely related with susceptibility of pomegranate to bacterial blight disease

Scientia Horticulturae 211 (2016) 79–86

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Nutrient imbalance indices are closely related with susceptibility of pomegranate to bacterial blight disease Ashis Maity a,∗ , Jyotsana Sharma a , Ananta Sarkar b , Amarja K. More a , R.K. Pal a a b

ICAR-National Research Centre on Pomegranate, NH-9, Kegaon, Solapur-413255, Maharashtra, India ICAR-Directorate of Research on Women in Agriculture, Bhubanewsar-751003, Odisha, India

a r t i c l e

i n f o

Article history: Received 15 February 2016 Received in revised form 17 August 2016 Accepted 19 August 2016 Available online 24 August 2016 Keywords: Pomegranate DRIS CND Nutrient imbalance indices Bacterial blight disease

a b s t r a c t Bacterial blight disease caused by Xanthomonas axonopodis pv. punicae is a major threat to pomegranate cultivation causing heavy losses. An attempt was made to establish nutrient sufficiency range for imparting moderate disease resistance and diagnosing nutrient imbalance causing disease development. Two approaches i.e. Diagnosis and Recommendation Integrated System (DRIS) and Compositional Nutrient Diagnosis (CND) were considered to evaluate the nutrient balance status in pomegranate. Nutritional survey revealed that concentration of Ca, Mg, Mn and Cu in leaves were significantly high in moderately resistant germplasms, while N, K and S concentration were observed to be high in susceptible ones. Deficiency of Ca was observed as the cause of disease in 80% of the susceptible germplasms studied. The next important nutrient deficiencies in the susceptible germplasm were Cu (77.14%), Fe (77.14%), Mn (68.57%) and Mg (65.71%). Nutrient imbalance indices of susceptible germplasm diagnosed through DRIS and CND had linear relationship (R2 = 0.93 and R2 = 0.82) with bacterial blight disease severity however, DRIS approach is superior for diagnosis of nutrient imbalances. As per DRIS analysis, leaf nutrient status of N 1.56–2.05%, P 0.11–0.28%, K 0.83–1.20%, Ca 1.60–2.16%, Mg 0.38–0.82%, S 0.09–0.16% and micronutrients viz. Fe 132.50–187.00 mg kg−1 , Mn 31.60–58.40 mg kg−1 , Zn 13.20–27.40 mg kg−1 and Cu 26.00–47.80 mg kg−1 could result in imparting moderate resistance to bacterial blight disease. The nutrient norms were validated and found to have linear relation between nutrient imbalance indices and bacterial blight disease severity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Pomegranate (Punica granatum L.), being an ancient medicinal fruit of tropical and subtropical regions of the world has emerged as an important commercial fruit in many Indian states including Maharashtra, Karnataka, Andhra Pradesh, Tamil Nadu, Gujarat, Rajasthan and Uttar Pradesh (Mondal and Mani, 2009). In the past, variety ‘Ganesh’ was predominantly cultivated in the state of Maharashtra, Karnataka and Andhra Pradesh. Recently improved variety such as ‘Bhagwa’ bred out of ‘Ganesh’ and ‘Gulesha Red’ was introduced in Maharashtra, Karnataka and Andhra Pradesh where it cover more than 90 per cent of cultivated area (Mondal and Singh, 2009).

∗ Corresponding author. E-mail addresses: [email protected] (A. Maity), [email protected] (J. Sharma), [email protected] (A. Sarkar), [email protected] (A.K. More), [email protected] (R.K. Pal). http://dx.doi.org/10.1016/j.scienta.2016.08.012 0304-4238/© 2016 Elsevier B.V. All rights reserved.

Intensive cultivation practices, lack of judicious nutrient management led to severe incidence of bacterial blight disease caused by Xanthomonas axonopodis pv. punicae (Xap), causing substantial damage to the pomegranate production and economic losses to the pomegranate growers (Mondal and Sharma, 2009). Various management options involving the application antibiotics, chemicals and cultural practices have been investigated. Treatment by only chemicals had limited success against the disease (Kumar et al., 2009). Presently, orchard health management (OHM) strategies were found to be effective to some extent. Improving plant resistance hold promise in OHM strategies for effective management of bacterial blight disease. Although plant disease resistance is genetically controlled (Agrios, 2005), it is affected by the environment and especially by nutrient deficiencies and toxicities (Marschner, 1995 Krauss, 1999). Information on nutrient status of diverse germplasm showing different degree of resistance to the disease could be utilized for developing nutrient norms for improving plant resistance against bacterial blight disease. Foliar analysis has frequently been used to be an important tool to monitor nutrient status of plant. Of several approaches, two

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approaches, the Diagnosis and Recommendation Integrated System (DRIS) (Walworth and Sumner, 1987) and Compositional Nutrient Diagnosis (CND) (Parent and Dafir, 1992) were established according to the principle of nutrient balance in plant and both of them could provide an approach to reflect the balance, sufficiency or deficiency of plant nutrients. DRIS and CND are based on the bivariate and multivariate diagnosis respectively (da Silva et al., 2004). These two approaches are more precise than sufficiency range approach (SRA). However, no attempt has been reported so far to diagnose the deficiency or toxicities of nutrient responsible for making plant susceptible to bacterial blight disease in pomegranate. The objectives of this research were: (i) to compare nutritional difference, if any, between moderately resistant germplasm and susceptible germplasm; (ii) to compare two different approaches (DRIS and CND) in diagnosis and evaluation of the nutrient status of pomegranate germplasm in relation to bacterial blight disease severity and in identifying the suitable form of these two approaches; (iii) to validate suitable approach in diagnosing nutrient status in relation to bacterial blight disease severity. 2. Materials and methods 2.1. Location and description The study consisted of two parts: (1) establishment of nutrient norms for improving resistance in plant against bacterial blight disease, (2) validation of nutrient norms in the farmers’ field. For the first part studied area was at pomegranate field gene bank, ICARNational Research Centre on Pomegranate research farm, Solapur, Maharashtra State, India located at 17◦ 48 N latitude and 75◦ 91 E longitude, at an altitude of 457 m above mean sea level. And the second part was conducted at pomegranate orchards of the most susceptible cv. Bhagwa located in three talukas i.e. Mohol, Sangola and Pandharpur of Solapur district, Maharashtra, India. The area under the study was semi-arid, showing hot summer and moderate winter with a mean annual maximum and minimum temperature of 40.4 ◦ C and 14.9 ◦ C, respectively and the average annual rainfall of 694 mm (approximately), occurring mostly during the months of July–September with 35–60 numbers of rainy days. The soil of the field gene bank at ICAR-NRCP, Solapur is loamy-skeletal Typic Ustorthents and properties are shown in Table 1. Orchard soils of Mohol, Sangola and Pandhar were loamy isohyperthermic Lithic Ustorthent. Soil characteristics of Mohol were: pH 8.0–8.4, free CaCO3 4.48–10.84%, organic carbon 0.83–1.59% and cation exchange capacity 14.6–17.8 cmol(p+ ) kg−1 soil and those of Sangola were: pH 7.7–8.1, free CaCO3 7.47–22.72%, organic carbon 0.57–1.29%, and cation exchange capacity 12.8–15.6 cmol(p+ )kg−1 soil while those of Pandharpur were: pH 7.7–8.2, free CaCO3 5.56–16.84%, organic carbon 1.08–1.82% and cation exchange capacity 18.8–20.5 cmol(p+ )kg−1 soil. 2.2. Experimental layout Sixty germplasm population comprising of IC318753, IC310790, IC318702, IC318712, IC318749, IC318740, IC1201, IC1203, IC318707, IC310728, IC318779, Accession no. 1, Accession no. 2, Accession no. 8, Accession no. 9, Accession no. 10, Accession no. 12, Accession no. 13, EC24686, EC104349, 1255, 1195, 1262, 1180, G526, P13, P16, P23, 17/2, Jodhpur collection, Double flower, Almoda, Bedanasuri, Kerala collection, Amlidana, Kalpitya, Yearcud HRS, Bedana thinsl, Bedanasri, Co-white, Gulesha red, Kabuli yellow, KRS, Pune collection, Sirin anar, Nimali, Dholka, Kasuri, Muskat, Spendanader, Bassain seedless, GR pink, Surat anar, Tabesta, Bas kalinsi, Dorsta, Spin sakaharir, Kabuli canoor, Damini, Bedanasedana was randomly planted in three blocks following

randomized complete block design (RCBD) in the Pomegranate field gene bank at ICAR-National Research Centre on Pomegranate showing varying degree of response to bacterial blight disease (differing degree of severity) were studied during July to November 2014 in the Mrigh bahar (rainy season flowering) which coincided the peak period of bacterial blight disease incidence. Four years old trees were selected for the investigation. Bacterial blight disease severity was assessed and recorded four times during the period. The plants were spaced at 4.5 m and 3.0 m in between and within the rows, respectively. The plants were provided with routine cultural practices suitable for commercial pomegranate production including pruning, defoliation and irrigation uniformly for Mrigh bahar crop (i.e. June-July flowering). One-third of the recommended dose of N-P2 O5 -K2 O (625-250-250 g per plant) along with 40 kg farmyard manure were applied immediately after the harvest of the previous crop and the remaining two-thirds of the recommended dose of N-P2 O5 -K2 O were applied in two equal splits, one at 45 days after flowering and other at 120 days after flowering. The source of N, P2 O5 and K2 O were urea, single super phosphate/di-ammonium phosphate, and sulphate of potash respectively. For validation of nutrient norms, seven pomegranate orchards cv. Bhagwa from each of the three talukas viz. Mohol, Sangola and Pandharpur were surveyed and bacterial blight disease severity were recorded with the collection of leaf samples.

2.3. Sampling and analysis In the nutritional status survey, the 8th leaf pair from the nonfruiting new flush was collected in field gene bank as well as in farmers’ orchards in Solapur district during August 2014 at flowering stage. The leaf samples were washed with tap water, deionized-water, 0.1 mol L−1 HCl and deionized-water (AlvarezFernandez et al., 2001). Then they were oven-dried at 65 ◦ C for 48 h to constant weight and grounded with agate mortar. The samples were digested with H2 SO4 to determine N and HNO3 /HClO4 (v/v, 4:1) to determine P, K, Ca, Mg, S, Fe, Mn, Zn and Cu. The leaf N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu were measured with the methods of AOAC (2005).

2.4. Establishment of DRIS parameters The calculation of DRIS is derived from Walworth and Sumner (1987). Field observation indicated that severity of disease on fruits is positively correlated with that on the foliage (r = 0.76*). It was also observed that bacterial blight disease severity above 20% causes considerable yield loss ranging from 30–80% depending on the severity of disease and prevailing weather condition and hence, 20% disease severity was considered as cut off value for separating moderately resistant and susceptible subpopulation (Singh et al., 2015; Anonymous, 2009). Bacterial blight disease severity on foliage and leaf nutrient concentration built a databank, which was divided to moderately resistant (disease severity ≤20%) and susceptible (disease severity >20%) sub-populations based on their impact on yield loss. Mean, standard deviation and variance were calculated for each subpopulation. The mean values (in moderately resistant subpopulation) of 10 nutrients expression were selected as the diagnostic norms for imparting moderate resistance to the pomegranate plant and their respective variance ratios is listed in Table 2. The DRIS norms established from the moderately resistant population of pomegranate germplasm were further employed to compute DRIS indices for the foliar mineral nutrient composition of susceptible germplasm and surveyed pomegranate orchards. DRIS

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Table 1 Soil properties of the moderately resistant plant and susceptible plant in Solapur, Maharashtra, India. Soil properties

Method followed

Moderately resistant germplasm (n = 25)

Susceptible germplasm (n = 35)

pH Available-N (mg kg−1 ) Available-P (mg kg−1 ) Available-K (mg kg−1 ) Available-Fe (mg kg−1 ) Available-Mn (mg kg−1 ) Available-Zn (mg kg−1 ) Available-Cu (mg kg−1 )

Soil water ratio of 1:2.5 Alkaline hydrolysis diffusion method Olsen’s method Extracted with 1 mol L−1 NH4 CH3 COO Extracted with DTPA extracting agenta Extracted with DTPA extracting agenta Extracted with DTPA extracting agenta Extracted with DTPA extracting agenta

8.22 121.63 4.50 241.57 10.59 25.77 16.17 19.78

8.19 138.90 5.76 363.42 11.04 22.97 12.20 18.12

a

DTPA extracting agent: 5 mmol L−1 DTPA, 0.01 mol L−1 CaCl2 and 0.1 mol L−1 TEA (triethanolamine).

Table 2 DRIS norms (means and CVa ) in pomegranate (Punica granatum L) for imparting moderate resistance against bacterial blight disease (caused by Xanthomonas axonopodis pv. punicae). DRIS norm

Mean

CV (%)

DRIS norm

Mean

CV (%)

DRIS norm

Mean

CV (%)

P/N N/Ca N/Mg N/Fe N/Mn N/Cu P/K P/Ca P/Mg P/Fe P/Mn P/Zn P/Cu K/Ca

0.10 1.02 3.31 1.18 × 10−2 0.05 0.05 0.19 0.10 0.33 1.15 × 10−3 4.50 × 10−3 9.94 × 10−3 5.20 × 10−3 0.53

22.84 10.81 20.87 13.04 15.78 17.99 27.10 29.42 34.22 24.19 29.55 22.25 30.85 11.94

K/Mg K/Fe K/Mn K/Cu Ca/Mg Fe/Ca Ca/Mn Cu/Ca Fe/Mg Mn/Mg Zn/Mg Cu/Mg Fe/Mn Fe/Cu

1.73 6.18 × 10−3 2.38 × 10−2 2.76 × 10−2 3.26 87.40 4.51 × 10−2 19.86 283.23 73.36 32.85 63.94 3.93 4.50

24.19 14.75 15.88 20.62 23.00 13.92 15.64 18.93 23.59 23.46 25.64 23.17 19.87 17.89

Zn/Mn Zn/Cu Cu/Mn S/N P/S S/K S/Ca S/Mg S/Fe S/Mn S/Zn S/Cu

0.46 0.52 0.89 5.98 × 10−2 1.65 0.12 0.06 0.20 7.10 × 10−4 2.74 × 10−3 6.14 × 10−3 3.13 × 10−3

23.29 18.26 19.36 13.09 25.28 16.01 17.88 24.66 18.30 17.45 17.06 15.50

a

CV means coefficient of variation (%).

Table 3 Leaf nutrient concentrations (means ± SDa ) of pomegranate (Punica granatum L.) for moderately resistant (n = 25) and susceptible germplasm (n = 35) in Solapur, Maharashtra, India. Item

Moderately resistant germplasm (n = 25)

N (%) P (%) K (%) Ca (%) Mg (%) S (%) Fe (mg kg−1 ) Mn (mg kg−1 ) Zn (mg kg−1 ) Cu (mg kg−1 )

1.88 0.18 0.98 1.85 0.59 0.11 160.04 41.93 18.64 36.32

a

± ± ± ± ± ± ± ± ± ±

Susceptible germplasm (n = 35)

0.15 0.05 0.10 0.14 0.13 0.02 16.02 7.55 3.13 5.44

1.98 0.22 1.15 1.53 0.48 0.14 136.21 33.82 19.19 29.33

± ± ± ± ± ± ± ± ± ±

0.15 0.11 0.17 0.31 0.16 0.04 26.85 10.77 2.05 12.76

P 0.0130 0.1411 <0.0001 <0.0001 0.0068 0.0005 0.0002 0.0020 0.4156 0.0127

SD means standard deviation, P means significance level at 95%.

nutrient index (IX ) of nutrient Xi was calculated for nutrient A-Z using Eqs. (1) and (2) (Beaufils, 1973).

 

















IA = f A/B + f A/C + f A/D + -------+f A/Z /n









































IB = −f A/B + f B/C + f B/D + -------+f B/Z /n

(1)

IZ = −f A/Z − f B/Z + f Z/D + -------+f Y/Z /n When A/B > a/b f (A/B) = [(A/B)/(a/b) − 1] ∗ 1000/CV

(2)

balances of nutrition; (iii) greater than zero means the nutrient X is excessive. NII M =

|IA | + |IB | + |IC | + --------+|IZ | n+1

(3)

The mean of Nutritional Imbalance Index (NIIM ) is the mean of the sum of absolute values of DRIS index (Eq. (3)); it is used to identify the nutrient imbalances. The higher value means the greater imbalance of plant nutrition.

When A/B < a/b f (A/B) = [1 − (a/b)/(A/B)] ∗ 1000/CV A/B is the ratio of two nutrient concentrations in samples for diagnosis, and a/b is the ratio of the standard, n is the number of the ratios, and CV is the coefficient of variation associated with each nutrient ratio norm a/b–a/z. The value of IX indicates the nutrient status of nutrient X, i.e. (i) less than zero means that the nutrient X is deficient for crop; (ii) equal to zero means the nutrient is in the

2.5. Establishment of CND parameters CND is a statistic-based model for individuals characterized by compositional data set (Parent and Dafir, 1992). The plant-tissue composition forms a d-dimensional nutrient arrangement, i.e. simplex (Sd ) made of d + 1 nutrient proportions including d nutrients and a filling value (Rd ) defined by Eqs. (4) and (5), where 1 is the

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dry matter concentration, N, P, K, etc. are nutrient concentrations (%) computed as follows: Sd = [(N, P, K...........Rd ) : N > 0, P > 0, K > 0, Rd > 0 N + P + K+, ..........., Rd = 1] R d = 1–(N + P + K + . . .. . ..)

(4) (5)

The nutrient proportions become scale invariant after they are divided by the geometric mean (G) of the d + 1 components including Rd (Aitchison, 1982). The formula can be expressed as Eq. (6). G = [NxPxKx. . .. . ..xR d ]1/d+1

Table 4 Nutritional status of susceptible pomegranate germplasm as assessed through DRIS and CND. Elements

Nitrogen Phosphorus Potassium Calcium

(6) Magnesium

VX is the CND row-centered log ratio for nutrient X, and calculated by Eqs. (7), and (8) is the calculation to ensure that VX has been calculated properly. According to Eq. (4), the sum of tissue component is 100%. Therefore, the sum of their row-centered log ratios, including the filling value (Rd ), should be zero. VN = ln(N/G), VP = ln(P/G), VK = ln(K/G), .........., VRd = ln(Rd /G) VN + VP + VK + ..............., +VRd = 0

(7) (8)

The VX functions are converted to CND nutrient index (IX ) by normalized calculation (Eq. (9)). IX value is a log linear contrast between two row-centered log ratio means and functions the same as DRIS. VN *, VP *, VK *,. . .. . .. . .,VRd * and SDN *, SDP *, SDK *,. . .. . ..,SDRd * are the CND norms as means and the standard deviations of row-centered log ratios of d nutrients, respectively. IN = (VN − VN ∗)/SDN ∗, IP = (VP − VP ∗)/SDP ∗

Iron Manganese Zinc Copper

Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess Deficient Sufficient/excess

DRIS

CND

17.00 82.85 20.00 80.00 11.43 88.57 80.00 20.00 65.71 34.29 8.57 91.43 77.14 22.86 68.57 31.43 42.86 57.14 77.14 22.86

20.00 80.00 14.29 85.71 11.43 88.57 88.57 11.43 68.57 31.43 8.57 91.43 77.14 22.86 74.29 25.71 22.86 77.14 80.00 20.00

germplasm (p < 0.05) whereas, the Ca, Mg, Fe, Mn and Cu concentrations were significantly higher (p < 0.05) in the moderately resistant germplasm than in the susceptible germplasm. 3.2. Diagnosis of nutrient balances in susceptible germplasm with DRIS

(9)

IK = (VK − VK ∗)/SDK ∗, IRd = (VRd − VRd ∗)/SDRd ∗ r2 is the mean of nutrient imbalances index of CND, calculated by Eq. (10). This index is used to evaluate the plant nutritional status; the higher the value, the greater the imbalance of the plant nutrition displays. r2 = IN 2 + IP 2 + IK 2 +, . . .. . .. . .. . .. . .., + IRd 2

Sulphur

Leaf nutritional status (%)

(10)

2.6. Statistical analysis Descriptive parameters (means, SD, CV and geometric mean), CND and DRIS norms were calculated by Microsoft Excel 2003. Oneway ANOVA was done using SAS software on significance analysis. Validation was carried out only in susceptible cultivars since there is no resistant cultivar grown commercially. 3. Results 3.1. The nutritional status of pomegranate germplasm Sixty pomegranate germplasm were divided into two subpopulations viz. moderately resistant sub-population and susceptible sub-population on the basis of bacterial blight disease severity. Twenty-five germplasm exhibiting upto 20% bacterial blight disease severity were grouped as moderate resistant germplasm and thirty-five germplasm showing more than 20% bacterial blight disease severity were grouped as susceptible germplasm during Mrigh bahar (July to December). The cut off value was selected 20% as bacterial blight disease above 20% causes considerable reduction in pomegranate yield and economic loss. The leaf nutrient concentrations of two groups of pomegranate germplasm are shown in Table 3. Leaf N, K, S concentrations were significantly higher in the susceptible germplasm than that in the moderately resistant

The DRIS norms were established with nutrient concentration data from the pomegranate germplasm (n = 60) in ICAR-NRC on Pomegranate field gene bank. The ratios of each two leaf nutrient concentrations of N, P, K, Ca, Mg, S (%), Fe, Mn, Zn and Cu (mg kg−1 ) were calculated and the coupled ratios (e.g. K/P and P/K) were selected by F-test and the ratio with higher F-value were selected as the best adequate expression (Walworth and Sumner, 1987). The DRIS norms established are listed in Table 2. Leaf nutrient concentrations in the susceptible pomegranate germplasm were evaluated with DRIS (Table 4). The nutrient sufficiency ranges for imparting moderate resistance in plant against bacterial blight disease derived from the DRIS norms were 1.56–2.05%, 0.11–0.28%, 0.83–1.20%, 1.60–2.16%, 0.38–0.82%, 0.09–0.16% for N, P, K, Ca, Mg and S and were 132.50–187.00, 31.60–58.40, 13.20–27.40 and 26.00–47.80 mg kg−1 for Fe, Mn, Zn and Cu, respectively. According to DRIS indices, 80.00%, 77.14%, 77.14%, 68.57% and 65.71% of the susceptible pomegranate germplasm were found deficient in Ca, Fe, Cu, Mn and Mg respectively. It was also observed that the germplasm with higher nutrient imbalance index (NIIM ) value i.e. higher nutrient imbalance recorded higher bacterial blight disease severity (Fig. 1). Significant linear relationship between DRIS nutrient imbalance indices and bacterial blight disease severity exist with R2 value as high as 0.93. The DRIS nutrient imbalance indices of the susceptible germplasm ranged from 63.86 to 432.14 with bacterial blight disease severity recorded varying from 21.67 to 66.67%. 3.3. Diagnosis of nutrient balances in susceptible germplasm with CND The leaf nutrient concentrations of the susceptible germplasm were also evaluated with the CND. The S10 i.e. ten-dimensional (10 + 1) pomegranate simplex comprised ten nutrients, N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu and Rd (the filling value). Nutrient concentrations were transformed into CND row-centered log ratios, VN * ,

A. Maity et al. / Scientia Horticulturae 211 (2016) 79–86

DRIS nutritional imbalance indices

Disease severity (%)

70

y = 0.0004x2 - 0.102x + 30.536 R² = 0.927

60 50 40 30 20 10

525

y = -0.002x2 + 2.793x + 35.98 R² = 0.981

450 375 300 225

150 75 0

0

0 0

100

200

300

400

500

DRIS nutrient imbalance index (NIIM) Fig. 1. Relation between DRIS nutrient imbalance index and blight disease severity in pomegranate germplasm. The R2 value 0.93 indicates that the fitted quadratic polynomial regression model explains about 93% of the total variation in the disease severity by DRIS nutrient imbalance index.

83

30

60

90

CND

120

150

180

r2

Fig. 3. Relation between CND r2 and DRIS nutritional imbalance indices in pomegranate germplasm. The R2 value 0.98 indicates that the fitted quadratic polynomial regression model explain about 98% of the total variation in the disease severity DRIS nutritional imbalance indices by CND r2 (nutrient imbalance indices).

Table 5 The CND norms (means ± SDa ) in pomegranate (Punica granatum L.) for imparting moderate resistance against bacterial blight disease (caused by Xanthomonas axonopodis pv. punicae). Mean ± SDa

CND norm

2.47 ± 0.06 0.12 ± 0.21 1.82 ± 0.10 2.45 ± 0.10 1.29 ± 0.18 −0.35 ± 0.11 −2.30 ± 0.11 −3.65 ± 0.13 −4.46 ± 0.12 −3.79 ± 0.14 6.39 ± 0.08 0.00

*

VN V* P V* K V* Ca V* Mg V* S V* Fe V* Mn V* Zn V* Cu * V Rd  V* x a

SD means standard deviation.

Disease severity (%)

70

y = 0.0018x2 - 0.11x + 26.039 R² = 0.8229

60 50

Fig. 4. Linear regression fit plot for disease severity on DRIS nutritional imbalance indices (DRIS NIIM ).

40 30 20 10 0 0

30

60

90

120

150

180

CND r2 Fig. 2. Relationship between CND r2 (nutrient imbalance index) and bacterial blight disease severity in pomegranate germplasm. The R2 value 0.82 indicates that the fitted quadratic polynomial regression model explain about 82% of the total variation in the disease severity by CND r2 (nutrient imbalance index).

VP * , VK * , VCa * , VMg * , VS * , VFe * , VMn * , VZn * , VCu * and VRd * through Eqs. (5)–(7). The CND nutrient indices (IX ) and the CND nutrient imbalance indices (r2 ) were calculated by Eqs. (9) and (10), respectively. The CND norms are shown in Table 5. The diagnosis results with the CND were consistent with that of the DRIS (Table 4). The nutrient imbalance indices (r2 ) of the susceptible pomegranate germplasm varied from 9.85 to 167.05 with bacterial blight disease severity recorded ranging from 21.67 to 66.67%. Significant linear relationship between CND nutrient imbalance indices (r2 ) and bacterial blight disease severity were recorded and is presented in Fig. 2 with R2 value 0.82. The nutritional status of the susceptible pomegranate germplasm diagnosed through CND was consistent with that

evaluated through DRIS with the exception of the Zn concentration (Table 4). Nutrient imbalance indices of both systems closely correlate, which is shown by the R2 (0.98) of the linear relationship between both (Fig. 3). However, we consider DRIS more suitable for the diagnosis of nutrient imbalances in relation to bacterial blight disease severity based on the higher R2 of the regression between the index and the disease severity compared to CND. 3.4. Validation of norms with a case study As observed above, the DRIS was better than CND in diagnosing nutrient imbalances with regard to bacterial blight disease severity. So, the DRIS approach was used to evaluate the nutritional status of twenty-one pomegranate cv. Bhagwa orchards from Mohol, Sangola and Pandharpur talukas of Solapur district, Maharashtra, India with the prevalence of bacterial blight disease severity 34.62–92.20%, 49.70–86.45% and 26.92–35.75% respectively. The DRIS nutrient imbalance indices showed linear relation with bacterial blight disease severity i.e. higher the nutrient imbalance indices, higher was the bacterial blight disease severity (Fig. 4). In all the orchards Mg, Ca and Mn were the most limiting nutrient and Cu was found limiting in 52.38% orchards which made plant susceptible to bacterial blight disease. The DRIS nutrient imbalance indices

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Table 6 DRIS nutrient balance indices and bacterial blight disease severity on leaves in three talukas of solapur district, Maharashtra, India.

were higher in Sangola and Mohol talukas compared to Pandharpur (Table 6). Accordingly, bacterial blight disease severity was significantly higher in Sangola and Mohol talukas as compared to Pandharpur. The leaf K concentrations was higher in pomegranate orchards of Sangola and Mohol while Mg and Mn concentrations was higher in pomegranate orchards of Pandharpur taluka registering lower bacterial blight disease severity (Figs. 5 and 6) compared to Sangola and Mohol talukas.

concentration of N in their leaves. Variation in nutrient status between the moderately resistant germplasm and the susceptible germplasm could be interpreted as genotypic effect leading to different nutrient uptake capacity (Tsipouridis and Thomidis, 2005; Jimenez et al., 2007) since phenotypic plasticity is often driven by nutrient supply, which was uniform for both groups of germplasm (Parent et al., 2013). Previous studies mentioned that different genotypes used different amounts of soil or fertilizer nutrients even if they were grown in the same condition (Kacar, 1995; Erdal et al., 2008). Different uptake was neither a result of the disease itself as there was no canker formation on the main stems, which generally disrupt the xylem vessel affecting the uptake of nutrients. Nutrients can affect the development of a disease by affecting plant physiology especially with respect to integrity of cell wall, membrane leakage and chemical composition of the host. Calcium is an important component of the cell wall structure as calcium poly galacturonates are required in the middle lamella for cell wall stability (Dordas, 2008). Magnesium is also a constituent of the middle lamella that along with Ca makes this pectic substance more resistant to degradation by pectolytic enzyme of pathogenic bacteria (Bateman, 1964; Bonner, 1950). Further, Mn controls lignin and suberin biosynthesis (Römheld and Marschner, 1991; Vidhyasekaran, 1997) through activation of several enzymes of the shikmic acid and phenyl propanoid pathways (Marschner, 1995). Both lignin and suberin are important biochemical barrier to pathogenic invasion (Kolattukudy et al., 1994; Rioux and Biggs 1994; Hammerschmidt and Nicholson, 2000; Vidhyasekaran, 1997, 2004). Although Fe does not affect lignin synthesis, it is a component of the peroxidase enzyme whose activity protect the plant cell from oxidative damage by detoxifying reactive oxygen species (ROS) generated at the site of infection (Wang et al., 2013). All these mechanisms in moderately resistant germplasm played important role in exhibiting resistance against bacterial blight disease. Several researchers could find that genetically engineered cotton, soybean and corn plants for herbicide tolerance were typically low in Mg and Mn and more susceptible to various diseases (Huber, 2010; Johal and Huber, 2009). Further at high N concentration in leaf, the metabolism of plant changes: as some key enzymes of phenol metabolism have lower activity, the content of the phenolics decreases and the lignin content may be lower- all these are part of the defense system of plant against infection. That is why, we could notice higher N concentration in leaf of susceptible germplasm. There is evidence that incidence of blight in pear trees increased with N and decreased with Mn fertilization (Koseoglu et al., 1996) that interacted with Mg. The higher K concentration in leaves indicates higher uptake of K, which made the plant susceptible to bacterial blight disease by limiting the uptake of Ca and Mg (Malvi, 2011), which plays a pivotal role in the defense mechanism of the plant against bacterial infection as discussed earlier. Sulphur (S) promotes ethylene biosynthesis (Yang and Hoffman, 1984; Yang, 1989; Kende, 1993). It has also been reported that ethylene plays an important role in disease development and there is clear correlation between ethylene production and pathogen induced tissue damage (Ben-David et al., 1986; Boller, 1991; Stall and Hall, 1984; Yang and Hoffman, 1984), which might be the reason for the higher S concentration in the leaves of susceptible germplasm in the present investigation.

4. Discussion

4.2. Comparison of diagnosis results with DRIS and CND

4.1. Nutritional status of pomegranate germplasm in relation to bacterial blight disease

In this research, both DRIS and CND can properly explain the variation of nutritional status in relation bacterial blight disease severity as we could observe linear relationship between DRIS NIIM vs. bacterial blight disease severity and CND r2 vs. bacterial blight disease severity. Nutritional status of susceptible germplasm as diagnosed through DRIS and CND have shown in Table 4. Although

Name of the Taluka

DRIS nutrient imbalance indices

Bacterial blight disease severity (%)

Mohol Sangola Pandharpur LSD at0.05

419.74b 503.80a 255.04c 78.26

60.15a 74.98a 31.26b 15.73

Different letters within each column indicate that the values are statistically significant by Duncan’s multiple range test (DMRT).

Fig. 5. Major nutrient concentration in leaves of pomegranate cv. Bhagwa of three talukas of Solapur district, Maharashtra, India. Vertical bars represents standard deviation of seven independent replicates. Different letters indicate that the values are statistically significant by Duncan’s multiple range test (DMRT).

Fig. 6. Micro-nutrient concentration in leaves of pomegranate cv. Bhagwa of three talukas of Solapur district, Maharashtra, India. Vertical bars represents standard deviation of seven independent replicates. Different letters indicate that the values are statistically significant by Duncan’s multiple range test (DMRT).

Moderately resistant germplasm were found to have higher leaf concentration of Ca, Mg, Fe and Mn than the susceptible germplasm. We could also notice that the susceptible germplasm had higher

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the extent of per cent gerplasm in deficient/sufficient or excess group varied slightly between two approaches viz. DRIS and CND, both the approaches identified the same most deficient nutrient and the least deficient nutrient. This suggests that the differences between the approaches may be minimal. Both the approaches suggested that susceptible germplasm were mostly deficient in Ca, Mg, Fe, Mn and Cu which are consistent with nutritional status survey of pomegranate germplasm (Table 3). Moreover, the relation between DRIS NIIM and CND r2 was linear and very close with high R2 value i.e. 0.98 which explain the robustness of the approaches in diagnosing nutrient imbalances with respect to their response to bacterial blight disease. Similar close relationship between DRIS and CND was reported by several researchers (Parent et al., 1993, 1994; Khiari et al., 2001). However, the R2 relationship between DRIS NIIM and bacterial blight disease severity had higher adjusted R2 (0.93) compared to the relationship between CND r2 and bacterial blight disease severity (0.82). This suggests that the DRIS approach may be superior in diagnosing nutrient imbalances with respect to their reaction to bacterial blight disease. Similar observation was also reported by Xu et al. (2015) while diagnosing nutritional status of apple orchards in Shaanxi Province of China for higher yield. The differences of the two approaches are: (i) DRIS and CND are based on the bivariate and multivariate diagnosis respectively, (ii) CND explains the concentration effect in crops when the filling value (Rd ) is defined in this approach. Therefore we can draw the conclusion that the calculation of bivariate and multivariate in the two approaches and the concentration effect in pomegranate plant can have less effect on the diagnosis results. Huang et al. (2012) drew the similar conclusion that DRIS and CND were suitable to diagnose and evaluate nutrient balances status of citrus trees grown on calcareous soil with high pH.

4.3. Validation of nutrient norms-case study Case study indicated that pomegranate orchards with higher nutrient imbalance indices recorded higher bacterial blight disease severity. Bacterial blight disease severity showed linear relation with DRIS nutrient imbalance indices (Table 6). Leaf nutrient status of pomrgranate orchards in three talukas revealed that orchards in Pandharpur taluka showed higher foliar concentration of Mg and Mn and lower concentration of N compared to other two talukas which helped in improving resistance in plant against invasion by bacterial pathogen Xanthomonas axonopodis pv. punicae, thereby recorded lower disease severity compared to other two talukas. The comparison of nutrient status among pomegranate orchards of Mohol, Sangola and Pandharpur talukas rightly corroborate the nutrient status of susceptible germplasm and moderately resistant germplasm. The results indicated that Mg, Ca and Mn deficiency in leaves rendered pomegranate plant susceptible to invasion by bacterial blight disease in Solapur district, Maharashtra, India. Most pomegranate growing areas are calcareous in nature. Supplementing soil with sufficient quantity of well decomposed organic manure will enhance the bioavailability of Ca to the plant through the production of HCO3 − . In addition foliar application of Mg(NO3 )2 and MnSO4 will enhance the concentration of Mg and Mn in the foliage. These nutrients in turn will reduce the susceptibility of the plant to bacterial blight disease. Of course excessive application of N should be avoided. Since there is no resistant commercial variety of pomegranate to bacterial blight disease available, adoption of proper nutrient management practices according to the requirement of crop will have far reaching consequences on the management of bacterial blight disease.

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5. Conclusion The results demonstrated that higher bacterial blight disease severity in pomegranate plant grown on high pH soil was mainly due to lower concentration of Mg, Ca, Mn and Cu and higher concentration of N in leaf. Two approaches (DRIS and CND) were used to diagnose the nutrient imbalances in relation to response of plant to bacterial blight disease, however, the DRIS approaches was more suitable than CND to diagnose and evaluate nutrient balances of pomegranate orchards grown on soil of high pH soil with respect to bacterial blight disease severity. The sufficiency ranges of foliar nutrient concentration for imparting moderate resistance in plant against bacterial blight disease are 1.56–2.05%, 0.11–0.28%, 0.83–1.20%, 1.60–2.16%, 0.38–0.82%, 0.09–0.16% for N, P, K, Ca, Mg and S and are 132.50–187.00, 31.60–58.40, 13.20–27.40 and 26.00–47.80 mg kg−1 for Fe, Mn, Zn and Cu respectively.

Acknowledgements This study was supported by the facility of pomegranate field gene bank which was established by Dr. Ram Chandra. The authors wish to thank Dr. Ram Chandra (Principal Scientist, Horticulture, National Research Centre on Pomegranate, Solapur, Maharashtra, India) for his sincere effort in establishing pomegranate field gene bank.

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