Indian Journal of Experimental Biology Vol. 57, April 2019, pp. 248-258

Phytophthora nicotianae Breda de Haan induced stress changes in rootstock genotypes

Kuldeep Singh1, RM Sharma1*, AK Dubey1, Deeba Kamil2, Lekshmy S3, OP Awasthi1 & GK Jha4 Division of 1Fruits & Horticultural Technology; 2Plant Pathology; 3Plant Physiology; and 4Agricultural Economics, ICAR-Indian Agricultural Research Institute, New Delhi (India)

Received 15 May 2017; revised 15 February 2018

Phytophthora spp. are the most serious threat to citrus industry worldwide. Being a soil borne problem, use of tolerant rootstocks is the most ecofriendly approach to manage the deadly diseases caused by this fungus. Here, we assessed the reaction of eight genotypes including sour , Troyer and six variants of C. jambhiri Lush. viz., RLC-5, RLC-6, RLC-7, Grambiri, rough and Italian against the inoculation of Phytophthora nicotianae. Inoculation of P. nicotianae infected the feeder roots of tested rootstocks to varying degree, expressing higher disease incidence (81.25%) and number of infected feeder roots (54.25-60.62%) depending on the rootstock. Troyer citrange and sour orange proved most tolerant rootstocks against the inoculated fungus. Phytophthora inoculation tended to increase - the levels of reactive oxygen species (H2O2 and O2 ), antioxidant enzymes (catalase, peroxidase, glutathione reductase, superoxide dismutase and β-1,3-glucanase) and protein content. However, it significantly reduced the levels of macro- (N, P, K Ca and Mg) and micro- (Cu and Zn) nutrients, although the extent of variation was rootstock specific. Overall, Troyer citrange and sour orange expressed the lowest variation in the levels of ROS, peroxidase (POX), superoxide dismutase (SOD) and β-1,3-glucanase, protein and nutrient contents, while rough lemon proved most strongly affected. Of the various variants of Citrus jambhiri, RLC-5 and Italian rough lemon proved more tolerant for Phytophthora nicotianae than rest of the clones tested.

Keywords: β-1,3-Glucanase, Biotic Stress, Fungal, Grambhiri, Italian rough lemon, Leaf nutrients, Reactive oxygen species, Rough lemon, , Sour orange, Troyer citrange

In spite of favourable soil and climatic conditions, causing serious losses in citrus industry throughout biotic and abiotic factors lower productivity of citrus the world. The attack of Phytophthora spp. may cause fruits in India1 is very low (12.08 t ha-1) as against the various symptoms including trunk cankers, and 32.79 t ha-1 in Indonesia2. Among the biotic factors, rots of the foot, crown, root and fruit7. P. nicotianae is Phytophthora species (fungal) are considered as one one of the most important soil-borne pathogens of of the most serious soil borne threats3 in the citrus which causes mortality of huge number of commercially citrus growing areas causing substantial trees8. The most serious disease caused by losses (10-30%) worldwide4, especially on susceptible Phytophthora spp. is foot rot (gummosis), which rootstocks5 like rough lemon (Citrus jambhiri Lush) persists on trunk for long time particularly in drier and Rangpur lime (C. limonia). In Central India, climatic conditions. The root infection by sizeable quantity of plants (>20%) die due to Phytophthora spp. causes root rot, however, when Phytophthora in citrus nurseries where 7-8 million infects aerial parts of the rootstock, the disease is 6 citrus plants are propagated annually . The disease known as foot rot or crown rot. Phytophthora spp. causes heavy destruction of the plantations, when infect the scion, just above the bud union, the and also reduces the life expectancy, quality and yield disease known as gummosis, causing necrosis in the potential of the trees in Punjab. inner bark and cambium of the trunk7. Phytophthora There are two main economically important spp. can also infect the root cortex resulting into Phytophthora species viz., Phytophthora nicotianae decay of feeder roots. In these small trees, loss of Breda de Haan (syn. P. parasitica) and P. citrophthora, significant numbers of roots can result in death of the ——————— tree. The uptake of water and mineral nutrient is *Correspondence: impaired, and the root carbohydrate reserves are Phone: +917840008856 10 E-mail: [email protected] depleted by the repeated attacks . SINGH et al.: PHYSIOLOGY OF CITRUS PLANTS IN PHYTOPHTHORA INOCULATION 249

Use of tolerant or resistant rootstocks is one of the national culture collection centre, Indian Type most ecofriendly and long term approach to manage Culture Collection, New Delhi for the confirmation Phytophthora induced stress. Many citrus rootstocks of its identity. have been found to express a variety of reactions to different kind of stresses7,11. Irrigation with saline Bulk soil inoculation with infected citrus roots The roots of these established diseased seedlings water also has direct effects on root pathogens. The were used as the source of disease inoculum14. Seven resistance of Phytophthora tolerant rootstocks can be months old nucellar seedlings of citrus rootstocks diminished significantly by the ability of 12 (Table 1) were planted in September, 2015 in the pots Phytophthora to tolerate high salinity . Hence, it is (12”) containing 8 kg sterilized soil mixture, which utmost important to identify suitable Phytophthora was inoculated with P. nicotianae through infected rot tolerant/resistant rootstock, and to understand the roots. One month after planting, 10 g mixture of urea, mechanism involved in the host and pathogen reaction single super phosphate and potassium sulfate in the for sustainable . Large genetic ratio of 1:1:1 was applied. The data were recorded diversity in citrus provides ample scope to select after 6 weeks of disease inoculation. Phytophthora tolerant/resistant genotypes. No systematic efforts have been made to evaluate Disease assessment indigenous variability to find out desirable Root rot incidence was calculated by dividing the Phytophthora tolerant reaction by citrus rootstocks. total number of diseased plants with total number of Keeping in view the severity and economic importance plants observed and multiplying with 100. The data of the diseases caused by Phytophthora fungus, as well on feeder root infection was recorded by dividing as the existing genetic diversity in citrus, we conducted number of feeder roots infected per plant with total this study to understand the reaction of different number of feeder roots per plant and multiplying with citrus rootstock genotypes against the inoculation 100. Brown colored infected feeder roots were of Phytophthora nicotianae. identified under binocular microscope and reconfirmed by the isolation of Phytophthora Materials and Methods nicotianae by same infected root. The present experiment was set at the experimental orchard of the Division of Fruits & Horticultural Biochemical studies Hydrogen peroxide (H O ) was estimated by Technology, IARI, New Delhi. The seeds of all the 2 2 forming titanium-hydro peroxide complex15. For this, rootstocks were sown in the nursery during first week 1.0 g titanium dioxide and 10 g potassium sulphate of December, 2014, and mulched with polyethylene were digested in 150 mL concentrated H SO for 4 h sheet till the end of January, 2015. The potting 2 4 over a hot plate, which was further diluted to 500 mL mixture (2 part soil: 1 part farm yard manure) was and stirred using a magnetic stirrer cum heater at sterilized with 1% formalin solution under a 70-80°C till making a clear transparent solution. The transparent polyethylene cover for 24 h. same solution was then diluted to 1.5 L and stored in 16 Maintenance of inoculum and citrus root inoculation dark brown bottle . with P. nicotianae Table 1 —Rootstock genotypes tested Pure culture of P. nicotianae was procured from Central Citrus Research Institute, Nagpur (India) and Rootstock Scientific name maintained on Potato Dextrose Agar medium at 4°C RLC-5 Citrus jambhiri Lush. (Accession No. for further use. The roots of citrus seedlings were IC 274698) inoculated with the pure culture of P. nicotianae by RLC-6 Citrus jambhiri Lush. (Accession No. IC 273847) spore suspension method. Soil inoculation was done RLC-7 Citrus jambhiri Lush. (Accession No. using a suspension of seven days old culture, for this IC 255451) purpose suspension of culture was prepared at the rate Grambhiri (GR) Citrus jambhiri Lush. 5 of 10 spores/mL. This suspension was added to the Italian rough lemon (IR) Citrus jambhiri Lush. 13 soil . The inoculated seedlings were periodically Rough lemon (RL) Citrus jambhiri Lush. checked for Phytophthora infection through isolation Sour orange (SO) Citrus aurantium L. of same pathogen from root of the seedlings. The Troyer citrange (TC) C. sinesnsis (L.) Osbeck x Poncirus pathogen was identified by sending the culture to trifoliata L.) Raf. 250 INDIAN J EXP BIOL, APRIL 2019

The leaf tissue (1.0 g) was ground to fine powder 15000 ×g for 20 min at 4°C. The obtained supernatant with the help of liquid nitrogen, followed by addition was strained through double layers of muslin cloth of 10 mL cooled acetone in a cold room (10°C). and stored at 20°C in refrigerator. This supernatant Mixture was filtered with Whatman No. 1 filter paper, was used as extract for the estimation of following and added with 4 mL titanium reagent and 5 mL antioxidant enzymes. ammonium solution to precipitate the titanium-hydro The activity of SOD in leaf sample was determined peroxide complex. The reaction mixture was according to method outlined by Dhindsa, Plumb & centrifuged for 10 min at 10000 rpm in a refrigerated Thorpe18. The assay was based on the ability of SOD centrifuge, and the precipitate obtained was dissolved to inhibit the photochemical reduction of nitro blue in 10 mL 2M H2SO4, and re-centrifuged thereafter. tetrazolium (NBT). The reaction mixture (3.0 mL) Supernatant was read at 415 nm against reagent blank was prepared in tubes consisting of 0.2 mL of 200 mM in UV-visible spectrophotometer (Specord 200, methionine, 0.2 mL of 3 mM EDTA, 0.1 mL of 2.25 mM Analytik Jena). Concentration of H2O2 was computed NBT, 0.1 mL of 60 µM ribloflavin, 100 µL of enzyme by referring to a standard curve prepared from known extract and phosphate buffer (100 mM; pH 7.8). The concentrations of H2O2. Standard curve of H2O2 was last component i.e. riboflavin was added, and the tubes prepared by taking a range of concentrations of H2O2, were agitated well. The reaction was initiated for a to which 10 mL of cold acetone were added, this was specified time of 15 min by keeping the tubes followed by various steps as in case of sample, and a 30 cm below a light bank consisting of two 15 Watt curve was drawn by plotting concentrations against fluorescent lamps. Lights were switched off after respective absorbance. 15 min, and immediately, the tubes were covered with -. The total superoxide radical (O2 ) content was a black cloth to stop the reaction. Thereafter, the assayed as per the method suggested by Chaitanya & absorbance of the mixture was read on UV-VIS double Naithani17. It is based on the principle of formation of beam PC 8 scanning Auto cell spectrophotometer blue coloured formazone by nitrobluetetrazolium (Specord 200, Analytik Jena) at 560 nm wavelength. A - chloride with O2 in the absence of total superoxide complete reaction mixture without enzyme extract, dismutase (SOD) activity. Plant tissue, homogenized developing maximum colour served as a control. The in pre-cooled phosphate buffer (0.2 M, pH 7.2) having absorbance (log A560) was plotted as a function of the 1.0 mM diethyl dithiocarbamate (for inhibiting SOD) volume of enzyme extract in the reaction mixture. The was centrifuged at 10000 rpm for 10 min in Sigma volume of enzyme extract resulting 50% reduction in refrigerated centrifuge (Model 3k 30 Osterode, the absorbance in comparison with the control was Germany), and immediately, the supernatant was read from the resultant graph. Each unit of SOD -. used for the estimation of O2 . The reaction mixture activity in the sample was considered as the amount (3.0 mL) was prepared in tubes consisting of of enzyme that caused 50% reduction in the 0.2 mL of 200 mM methionine, 0.1 mL of absorbance as compared to the control tube lacking 3 mM EDTA, 0.1 mL of 2.25 mM NBT, 50 µL of enzyme extract. SOD was finally quantified on the 1.5 M sodium carbonate, 250 µL of enzyme extract basis of content of soluble protein the sample. and 2.30 mL water. It was incubated for 10-15 min at The CAT assay was based on the absorbance of 30°C, and absorbance was recorded at 540 nm on H2O2 at 240 nm in UV-range. A decrease in UV-VIS spectrophotometer (Specord 200, Analytik absorbance was recorded over a time period19. A Jena). The superoxide content was expressed as reaction mixture (3.0 mL) which consisted of 1.5 mL -1 -1 Δ540 Mm g FW. of 100 mM potassium phosphate buffer (pH 7.0), For preparation the enzyme extract, the fresh leaves 0.5 mL of 75 mM H2O2, and 200 µL of enzyme from each replication were collected during extract was prepared in test tubes and rest water to December in ice box and washed in running tap water make up the volume. On addition of H2O2, instantly followed by double-distilled water. The cleaned leaf the reaction started and decrease in absorbance was sample (1.0 g) was homogenized in pre-chilled mortar observed for 1.0 min at 240 nm. CAT activity was and pestle with liquid nitrogen followed by adding quantified as concentration of H2O2 reduced (initial 10 mL of chilled phosphate buffer (100 mM; pH 7.5) readingfinal reading = quantity of H2O2 reduced) -1 containing 0.5 mM EDTA. The homogenate and expressed as μmoles H2O2 hydrolysed mg -1 was collected in oak-ridge tubes and centrifuged at TSP min . SINGH et al.: PHYSIOLOGY OF CITRUS PLANTS IN PHYTOPHTHORA INOCULATION 251

The method suggested by Thomas, Jen & Morr20 by 0.2% Teepol solution and 0.1NHCl and double- was used to estimate POX activity in leaf sample. The distilled water. The washed leaves were packed in assay guaiacol utilized as the enzyme substrate. The paper bags, and kept for drying in a hot air oven at enzyme extract was prepared by homogenizing 1.0 g temperature of 70±2°C. After drying, leaf samples of clean leaf sample. The reaction mixture was were ground with the help of a Wiley mill and then prepared in tubes by adding 3 mL of phosphate buffer sieved through 1.0 mm mesh sieve. Ground leaf (0.1 M; pH 6.1), 500 µL of H2O2 (12 mM), 500 µL of samples were digested in diacid by using HNO3 and guaiacol (96 mM) as enzyme substrate and 200 µL of HClO4 in the ratio of 9:4. The digested leaf tissue was enzyme extract. The absorbance was read at 470 nm then diluted and filtered using Whatman No.1 filter wavelength on UV-VIS double beam PC 8 scanning paper in series. Thereafter double distilled water was Auto cell spectrophotometer, Specord 200, Analytik added to make final volume 100 ml. This diluted Jena. An increase in absorbance was observed at 30 s material was used for the determination of mineral intervals till the constant reading. nutrients viz., potassium (K), calcium (Ca), The powder of frozen root tissue, ground at 4C in magnesium (Mg), iron (Fe) and copper (Cu). an ice-chilled mortar with liquid nitrogen was suspended in 100 mM MacIlvaine (/ Tissue nutrients analysis Nitrogen (N) content in leaves was determined by NA HPO ) extracting buffer, pH 6.8 (1:1, w/v). 2 4 using Digestion Block method23. For this, finely Crude homogenate was centrifuged (15000 ×g) at 4C ground leaf tissue (1.0 g) was taken in digestion tube, for 30 min, and the supernatant fractions were kept and added with concentrated H2SO4 (6.0 mL). Then frozen at 20C. digestion tubes were then attached to digestion Total β-1,3-glucanase was assayed colorimetrically 21 system, and heated for about 385°C. The digestion using the laminarin dinitrosalicylic method . The was allowed to continue till the disappearance of reaction mixture consisting of 1.0 mg mL-1 laminarin -1 black or brown colour. The digestion tubes were then and 0.5 mL of 5 mg mL β-1, 3-glucanase (dissolved put to distillation unit which was set up to perform the in 50 mM sodium-phosphate buffer, pH 6.0) was o various steps like dilution, addition of alkali, steam incubated for 30 min at 50 C, thereafter, 1.0 mL DNS generation and titration. About 25.0 mL of boric acid reagent was added, and tubes were placed in boiling (4%) containing mixed indicator in a 250 mL conical water bath for 10 min, cooled, and added with 4.0 mL flask and kept it under ammonia receiving tube of the of distilled water. The amount of liberated reducing distillation assembly and distillation ran for 2.5 min. sugar was measured at 540 nm. One unit of enzyme The distillate was then titrated against 0.1N H2SO4 activity was defined as the amount of enzyme that until the appearance of purple colour. Nitrogen in leaf catalyzed the liberation of reducing sugar equivalent samples was determined by using following formula; to 1.0 µg D-glucose per min under standard assay conditions. N (%) = [(T-B) × N × 1.4] /S

Soluble protein content was estimated using where, T = Volume of standard H2SO4 taken for 22 Bradford protein assay . It was based on the principle sample; B = Volume of standard H2SO4 taken for the maximum absorbance for an acidic solution of blank; N = Normality of acid; S = Weight of plant Coomassie Brilliant blue G250 shifts after binding sample taken with protein from 465 to 595 nm. A standard curve The content of phosphorus (P) in leaves was was prepared using bovine serum albumin (BSA) determined by vando-molybdo-phosphoric yellow stock of 100 μg/ mL followed by dilutions from this colour method23. In view of that, 0, 1, 2, 3, 4 and 5 mL stock solution in the range of 0 to 100 μg/ mL were of standard solution was transferred to volumetric taken to prepare a standard curve. Bradford reagent flask (50 mL ) to get 0, 1, 2, 3, 4 and 5 ppm of P, (2 mL) was added to standard BSA. It was incubated respectively, and in each flask, 10 mL of vando- at room temperature (20°C) for 10 min in dark. molybdate solution was added. The volume was made Absorbance was read at 595 nm in UV-Visible up with distilled water, thereafter the transmittance spectrophotometer (Specord 200, Analytik Jena). was recorded on spectrophotometer (UV-VIS double From the each replication, 20 leaves were collected beam PC 8 scanning auto cell spectrophotometer for determination of leaf nutrients concentration. The (UVD-3200, Labomed, Inc., Culver city, USA) after collected leaves were washed in tap water followed 30 min at 420 nm wavelength. Then the absorbance 252 INDIAN J EXP BIOL, APRIL 2019

was plotted against concentration. The concentration sour orange and Troyer citrange rootstocks to have of P was calculated by using the standard curve. the resistance to root rot and gummosis caused by P in sample (%) = A/100×V Phytophthora species. Mohammed, Belmehdi & Zemzami27 also reported the Troyer citrange to have a where, A = P concentration in µg as read against good degree of resistance similar to sour orange, sample reading on the standard curve and V = Volume whereas rough lemon and Rangpur lime proved of aliquot taken (mL) for colour development. highly susceptible against Phytophthora species. Total leaf potassium (K) content was estimated Cheema, Dhillon & Kapur28 suggested that the degree from diacid digested leaf samples using a of tolerance/ resistance to Phytophthora spp. of microprocessor based flame photometer (Flame different rootstocks could be arranged as Kinnow Photometer-128, Systronics, Ahmedabad). Calcium, (least resistant) 50%. RLC-5, RLC-6 and Italian rough lemon

Statistical analysis appeared similar to sour orange, but Troyer citrange The experiment was laid out in a Completely proved much better exhibiting lowest feeder root rot Randomized Design (CRD) with four replications, infection. Certain rootstocks have been reported having ten plants per replication. Data were analysed resistant because roots become infected but do not rot, using the SAS package (9.3 SAS Institute, Inc, and while others are classified as tolerant because they USA) to calculate F values followed by Tukey's Table 2 — Influence of P. nicotianae on disease incidence and honest significance test. P values ≤0.05 were infected feeder roots of citrus rootstocks considered as significant. Treatment Disease incidence Feeder root (%) infected (%) Results and Discussion Non-inoculated The disease incidence caused by Phytophthora was c e significantly affected in various rootstock genotypes RLC-5 0.00 (0.71 )* 0.00 (0.71 ) c e (Table 2). Grambhiri and rough lemon showed the RLC-6 0.00 (0.71 ) 0.00 (0.71 ) c e highest disease incidence followed by RLC-5, RLC-6, RLC-7 0.00 (0.71 ) 0.00 (0.71 ) c e RLC-7 and Italian rough, sour orange and Troyer Grambhiri 0.00 (0.71 ) 0.00 (0.71 ) citrange. Similar to disease incidence, the numbers of Italian rough lemon 0.00 (0.71c) 0.00 (0.71e) infected feeder roots were highest in rough lemon Rough lemon 0.00 (0.71c) 0.00 (0.71e) followed by Grambhiri and RLC-7. Percentage of Sour orange 0.00 (0.71c) 0.00 (0.71e) feeder root infection in RLC-5, RLC-6 and Italian Troyer citrange 0.00 (0.71c) 0.00 (0.71e) rough ranged between 33.26 and 34.03%. Sour orange Phytophthora inoculated and Troyer citrange showed significantly low feeder RLC-5 62.50 (7.90a) 34.03 (5.75cd) root infection, although these were statistically similar RLC-6 62.50 (7.90a) 33.26 (5.77cd) with RLC-5 and RLC-6. RLC-7 62.50 ( 7.90a) 43.90 (6.61b) Young generating roots of citrus rootstocks Grambhiri 81.25 (9.02a) 54.25 (7.39a) generally are highly susceptible to infection by biotic Italian rough lemon 62.50 (7.90a) 34.15 (6.04cb) stresses25. In the present study, rootstock genotypes Rough lemon 81.25 (9.02a) 60.62 (7.84a) had varying levels of disease incidence and number of b cd feeder roots infected with P. nicotianae. Sour orange Sour orange 37.50 (6.08 ) 30.24 (5.53 ) b d and Troyer citrange had <50% disease incidence, Troyer citrange 31.25 (5.56 ) 23.87 (5.08 ) which indicated tolerance power of these rootstocks, LSD (P ≤0.05) while all the strains of C. jambhri (RLC-5, RLC-6, Phytophthora (P) 0.32 0.13 RLC-7, Grambihri, rough lemon and Italian rough Rootstock ( R) 0.64 0.27 lemon) had >50% disease incidence, being highest in P × R 1.63 0.69 rough lemon. Our study confirms the findings of [Values are representing different letters are significant at P ≤0.05 Matheron, Wright & Porchas26, who had reported the (THST). *Values in parenthesis are square root transformed values]

SINGH et al.: PHYSIOLOGY OF CITRUS PLANTS IN PHYTOPHTHORA INOCULATION 253

generate new roots to maintain root mass density in (9.09%), sour orange (10.52%) and Italian rough Phytophthora infected soils. Young fibrous roots of (16.67%) than other rootstocks tested. most rootstocks support equally high populations of Reactive oxygen species (ROS like superoxide, Phytophthora spp. However, as roots of resistant or hydrogen peroxide etc.) are produced in many ways in tolerant rootstocks age, the pathogen population several cellular compartments, during normal declines in rhizosphere soil, whereas populations are metabolic processes30. Mitochondrias not only are a sustained on susceptible rootstocks29. source of reactive oxygen species (ROS) but also are The levels of ROS namely hydrogen peroxide sites of oxidative damage. High ROS levels are -. (H2O2) and superoxide radicals (O2 ) in the leaves of further enhanced during stresses which may cause the rootstock genotypes were significantly influenced, mitochondrial damage, and lead to induction of and tended to be increased at varying degree by programmed cell death31. In the present study, lower Phytophthora inoculation (Table 3). Phytophthora increase in H2O2 (<15%) was recorded in Troyer inoculated seedlings of rough lemon expressed the citrange, sour orange and RLC-5, being lowest in -. highest increase of H2O2 (52.43%), and least Troyer citrange. Similarly, O2 generation was also upregulation in H2O2 was measured in the leaves of lower in Troyer citrange, sour orange and Italian Troyer citrange (4.33%) as compared to the respective rough lemon. Phytophthora inoculation tends to -. control. However, the highest increase in O2 radicals increase several ROS in the leaves of infected due to Phytophthora infection was noticed in the plants32. However, stress resistant rootstocks have -. leaves of rough lemon (46.15%) followed by been reported to have less production of H2O2 and O2 Grambhiri (46.00%), RLC-7 (38.00%), RLC-6 in leaves as well as roots than that of susceptible (26.67%) and RLC-5 ( 25.00 %). The increase in the rootstocks33. Resistant rootstocks maintain their -. levels of O2 radicals was lower in Troyer citrange structural cell integrity under stress, producing -. smaller increases in the levels of H2O2 and O2 , and Table 3—Influence of P. nicotianae on reactive oxygen species MDA34, which increases largely in susceptible level in citrus rootstocks rootstocks, as also been observed higher in rough H O (mmoles of O -. Treatment 2 2 2 lemon and RLC-6 during the course of present study. H O mg-1 FW) (Δ Mm-1 g-1 FW) 2 2 540 Phytophthora inoculation significantly increased Non-inoculated the activity of antioxidant enzymes catalase (CAT), egf -4c RLC-5 157.39 16×10 peroxidase (POX) and superoxide dismutase (SOD) in RLC-6 142.35hgf 15×10-4dc hgf -4de leaves (Table 4) and ß-1,3-glucanase in roots (Fig. 1A) RLC-7 144.09 13×10 of different rootstocks at varying levels. Phytophthora Grambhiri 136.48hg 13×10-4de h -4b inoculation tended to show the higher upregulation of Italian rough lemon 118.82 18×10 CAT activity in Troyer, Grambhiri, RLC-7 and Italian Rough lemon 142.48hgf 13×10-4ed bdg -4ba rough (41.54-45.90%) as compared to their respective Sour orange 196.28 19×10 control. However, it was poorly upregulated in RLC-5, Troyer citrange 197.44bac 11×10-4f RLC-6 and rough lemon rootstocks (0.85-7.28%). Phytophthora inoculated Of the eight rootstock genotypes, highest edc -4ba RLC-5 174.81 20×10 upregulation in the activity of POX was observed in a -4ba RLC-6 217.50 19×10 Phytophthora inoculated seedlings of RLC-5(48.60%) RLC-7 186.43bdc 18×10-4b edf -4ba followed by sour orange (34.59%), RLC-7 (31.38%), Grambhiri 167.22 19×10 rough lemon (30.58%), Grambhiri (30.53%) and Italian rough lemon 156.91egf 21×10-4ba a -4ba RLC-6 (30.22%), while it was lowest in Troyer Rough lemon 217.79 19×10 citrange (18.40%) as compared to its non-inoculated Sour orange 215.62a 21×10-4a ba -4fe seedlings. Troyer citrange 206.21 12×10 Like other antioxidant enzymes, SOD activity LSD (P ≤0.05) was also upregulated in Phytophthora inoculated -5 Phytophthora (P) 5.16 4.2×10 seedlings of the rootstocks tested. The highest -4 Rootstock (R) 10.32 1.0×10 increase in SOD activity was found in Italian -3 P × R 26.27 2.0×10 rough (38.34%), and it was lower in sour orange and [Values are representing different letters are significant at P ≤0.05 (THST)] Troyer citrange (8.15-9.95 %) as compared to their

254 INDIAN J EXP BIOL, APRIL 2019

Table 4—Influence of P. nicotianae on antioxidant enzymes activity and protein content in the leaves of citrus rootstocks CAT (μmoles H O hydrolysed mg–1 POX (Unit µmol tetra-guiacol mg–1 SOD Treatment 2 2 TSP min–1) TSP min–1) (Unit mg–1 TSP min–1) Non-inoculated RLC-5 3.72cb 63.63ef 14.75bdc RLC-6 3.49cd 67.62efd 14.55dc RLC-7 3.29ed 16.17h 11.34egf Grambhiri 3.06ef 56.63efg 12.66edf Italian rough lemon 1.83h 70.35ed 10.30hgf Rough lemon 3.43cd 36.42hg 14.79bdc Sour orange 2.75gf 69.41efd 17.65ba Troyer citrange 2.72g 105.45ba 7.84h Phytophthora inoculated RLC-5 3.85b 94.55bc 16.48bac RLC-6 3.52cd 88.06bcd 17.80a RLC-7 4.49a 21.24h 14.55dc Grambhiri 4.34a 73.92ecd 16.68bac Italian rough lemon 2.67g 86.65bcd 14.25edc Rough lemon 3.68cb 47.56fg 16.37bac Sour orange 3.06ef 93.42bc 19.09a Troyer citrange 3.85b 124.86a 8.62hg LSD (P ≤0.05) Phytophthora (P) 0.06 4.44 0.59 Rootstock (R) 0.12 8.87 1.18 P × R 0.31 22.6 3.02 [Values are representing different letters are significant at P ≤0.05 (THST)]

Fig. 1 — Influence of Phytophthora nicotianae inoculation on the (A) activity of B1, 3 glucanase; and (B) protein content in citrus rootstock genotypes respective controls than other genotypes of citrus sour orange, rough lemon and RLC-5, while the rootstocks tested. similar response in respect of CAT was registered in In order to cope with this stress, plants evolve some sour orange, RLC-5, RLC-6 and rough lemon mechanisms called antioxidative system35 to detoxify seedlings. However, POX activity measured quite these oxidative chemicals such as SOD, CAT and lower in Troyer citrange and Italian rough lemon than POX enzymes36-37. During the course of present study, their respective control. Results of present study lower increase in the activity of SOD was noticed in showed that Troyer citrange and sour orange -. Phytophthora inoculated seedlings of Troyer citrange, rootstock genotypes produced lower H2O2 and O2 as SINGH et al.: PHYSIOLOGY OF CITRUS PLANTS IN PHYTOPHTHORA INOCULATION 255

well SOD activity, strongly supports the lower Pineapple sweet orange had also been reported by requirement of antioxidant defence system. However, Albrecht and Bowman14. rootstock genotypes such as Italian rough lemon also An upregulated level of protein was measured in increased higher CAT, POX and SOD could help to Phytophthora inoculated seedlings compared to non- bring down ROS levels in these rootstocks. Higher inoculated seedlings of all the rootstock genotypes. Of increase in POX and CAT activities in Italian rough the various combinations of Phytophthora inoculation lemon and RLC-5 might be the reason of lower levels and rootstocks genotypes, maximum protein content of ROS in the leaves of these rootstocks. It is also was registered in the Phytophthora inoculated pertinent to mention that rootstock genotypes RLC-6, seedlings of Troyer citrange (Fig. 1B). Phytophthora Grambhiri and rough lemon appeared to be worst inoculation showed the highest increase in protein rootstocks genotypes as they had higher ROS content in the leaves of rough lemon (56.40%), while generation with low level of antioxidant enzymes. in RLC-5, sour orange and Troyer citrange, it was Reports on variation in antioxidant enzymes activities quite lower (13.33-14.40 %) than rest of the caused by rootstock genotypes exist for different rootstocks tested, as compared to their respective citrus rootstocks37,38. In apple, Wang, Liang, Li, Hao, control. Of the various rootstock genotypes, Troyer Ma & Shu34 also observed the higher activities citrange tended to maintain the highest level of of SOD, POX and GR in Malus prunifolia than soluble protein followed by sour orange with or M. hupenhensis in response to stress. Hence, without fungal inoculation. Highest level of soluble rootstock former exhibited a strong protective protein has also been reported in Troyer citrange37. mechanism due to better maintained structural Albrecht & Bowman14 recorded higher leaf protein in integrity during exposure to stress than later Shun Chu Sha, Cleopatra mandarin and US 897 than rootstock. Boava, Cristofani-Yaly, Stuart & other rootstocks, while infected with P. palmivora. Machado39 suggested involvement of several proteins Phytophthora inoculated seedlings of RLC-5, with higher levels, including B1, 3-endoglucanase, RLC-6, RLC-7, Italian rough lemon, Grambhiri chalcon synthase, lipoxygenases and peroxidases in and rough lemon (all C. jambhiri) had significantly the resistant interaction between Poncirus trifoliata lower N, P and Cu leaf content as compared to their and P. nicotianae. non-inoculated seedlings, while no significant change Highest increase of β 1,3 glucanase activity in the in these nutrients content was observed in sour orange roots of inoculated seedlings of Troyer citrange and and Troyer citrange rootstocks following fungal sour orange was observed and have non-significant inoculation (Table 4). Similarly, Phytophthora differences with inoculated seedlings of RLC-5 and treatment failed to have any influence on K and Ca Italian rough and untreated seedlings of Troyer leaf content in either of the rootstock genotypes citrange (Fig. 1A). Non-inoculated seedlings of (Table 5). Moreover, leaf Mg content decreased RLC-7, Grambhiri, Italian rough lemon and rough significantly in treated seedlings as compared to lemon had the lowest activity of β-1,3-glucanase control in most of the rootstock except sour orange. In (0.010 IU mg-1 in each) without showing significant other hand, leaf Fe content decreased in all the difference with non-inoculated seedlings of RLC-6 rootstock genotypes under Phytophthora inoculated and inoculated seedlings of RLC-7 and Grambhiri except RLC-7 as compared to their respective control rootstocks. Compared to the respective non- seedlings. inoculated seedlings, Phytophthora inoculation Phytophthora inoculation tends to reduce the resulted the highest increase in β-1,3-glucanase length and number of feeder roots32, causing activity in Italian rough lemon (40.00%) followed by generalized dysfunction in water relations, reducing RLC-6 (30.00%) and RLC-5 (16.70%). However, it root hydraulic conductivity, and uptake of nutrients40. was lowest in Troyer citrange (7.14%) rootstock. In In the present study, it was also found that most of the the present study, higher β-1,3-glucanase activity macro- and micro-nutrients decreased in leaf tissues were observed in the roots of Troyer citrange, sour of susceptible rootstock genotypes. However, orange, Italian rough lemon and RLC-5 expressed rootstock genotypes expressed lower generation of their tolerance against P. nicotianae. Similar activity ROS (sour orange and Troyer citrange), and could of β-1,3-glucanase in the roots of sour orange and maintain leaf nutrient concentration even under 256 INDIAN J EXP BIOL, APRIL 2019

Table 5—Influence of P. nicotianae on leaf nutrient status of citrus rootstocks Treatment N (%) P (%) K (%) Ca (%) Mg (%) Cu (ppm) Fe (ppm) Non-inoculated RLC-5 2.64a 0.13ef 0.72ba 2.81b 0.27cb 15.25a 172.03b RLC-6 1.58e 0.14ed 0.73ba 2.75cebd 0.25ced 12.50bc 170.25cd RLC-7 1.92d 0.17bac 0.68bac 2.76cbd 0.26cbd 12.50bc 168.25fe Grambhiri 1.98dc 0.18a 0.68bac 2.71cefd 0.26cbd 13.75ba 167.25f Italian rough lemon 2.07c 0.16bc 0.73ba 2.78cb 0.28b 14.00ba 171.00cb Rough lemon 2.24b 0.16dc 0.70ba 2.75cebd 0.24ed 14.00ba 168.50fde Sour orange 2.32b 0.18a 0.76a 3.53a 0.35a 12.00bcd 175.16a Troyer citrange 2.35b 0.17bac 0.61edc 2.57gh 0.23fe 8.00gf 160.03h Phytophthora inoculated RLC-5 2.02dc 0.12f 0.67bac 2.77cbd 0.24ed 10.86ecd 170.14cd RLC-6 1.53e 0.13ef 0.65bdc 2.70efd 0.21fgh 9.11gef 167.21f RLC-7 1.65e 0.15dc 0.61edc 2.72cefd 0.21fgh 9.66gefd 167.19f Grambhiri 1.64e 0.16bdc 0.61edc 2.67ef 0.21fg 9.79gefcd 163.00g Italian rough lemon 1.64e 0.13ef 0.67bdc 2.76cbd 0.25ced 12.11bcd 169.50cde Rough lemon 1.53e 0.12f 0.58ed 2.64gf 0.18h 9.07gef 162.00g Sour orange 2.27b 0.18a 0.73ba 3.49a 0.33a 10.33efcd 170.50cb Troyer citrange 2.26b 0.16bdc 0.55e 2.53h 0.20gh 7.12g 158.14i LSD (P ≤0.05) Phytophthora (P) 0.02 0.003 0.02 0.02 0.005 0.53 0.34 Rootstock ( R) 0.05 0.01 0.03 0.03 0.010 1.06 0.69 P × R 0.13 0.02 0.09 0.08 0.025 2.72 1.75 [Values are representing different letters are significant at P ≤ 0.05 (THST)]

Phytophthora induced stress condition. Sour orange attacks the unsuberized roots (roots without and Troyer citrange could able to maintain N, P, K, deposition of suberin) making dysfunctional to the Ca and Cu content even under fungal stress. primary organs, responsible for the uptake of water Sour orange also maintained Mg level, despite of and mineral nutrients. The decay of roots soon leads Phytophthora inoculation. From the data of present to drought stress in the affected trees, which leads to study, it is clear that all the rootstocks showed nutrient depletion due to reduced surfaces of reduction in leaf Fe content under Phytophthora absorption and failure of tree water transport induced stress except RLC-7. Overall, Phytophthora systems44. The series of events like stronger inoculation tended to reduce the highest reduction in modulation of a number of genes implicated in N, P, K, Ca, Mg, Cu ad Fe in the seedlings pathogen perception, signal transduction, HR-like of rough lemon. Rough lemon has also been response, transcriptional reprogramming, hormone reported to be a poor P, Zn and Cu accumulating signalling and cell wall modifications have been rootstock41. Stolzy, Labanauska, Klotz & Dewolfe42 reported vital to a successful defense strategy in the also observed the significant decrease in P, Ca tolerant rootstock (sour orange) as compared to the and Fe in lemon, while grown in soil infected susceptible rootstock (sweet orange)45. with Phytophthora spp. Root rot also occurs on Results of this study indicate that the tolerance susceptible rootstocks in fruit-bearing groves where power of citrus rootstocks genotypes against damage rarely kills the tree, but the tree declines in Phytophthora can be confirmed on the basis of ROS vigour and fruit production. The repeated attacks of and β-1,3-glucanase levels in leaves and root tissues. Phytophthora tend to impair the uptake of water and Lower ROS levels and higher activities of β-1,3- mineral nutrients10, and deplete the carbohydrate glucanase, POX and CAT activity can be correlated reserves in the roots43. Phytophthora caused root rot with higher tolerance against P. nicotianae induced reduces leaf concentration of N, P, S, Zn and B to stress. Of the various rootstocks tested, Troyer below critical values for optimum growth, as it citrange, sour orange, RLC-5 and Italian rough lemon SINGH et al.: PHYSIOLOGY OF CITRUS PLANTS IN PHYTOPHTHORA INOCULATION 257

can be considered as tolerant rootstocks against the 17. Chaitanya KSK & Naithani SC, Role of superoxide, lipid Phytophthora nicotianae. peroxidation and superoxide dismutase in membrane perturbation during loss of viability in seeds of Shorea robusta Gaertn. f. New Phytol, 126 (1994) 623. References 18. Dhindsa RS, Plumb DP & Thorpe TA, Leaf senescence 1. Anonymous, Horticultural statistics at a glance. Ministry of and lipid peroxidation. Effect of some phyto-hormones and Agriculture & Farmers Welfare, Department of Agriculture scavengers of free radicals and singlet oxygen. Physiol and Farmers Welfare, Government of India. (2017) 15. Plant, 56 (1981) 453. 2. https://knoema.com 19. Aebi H, Catalase in vitro. In: Methods in Enzymology (Ed. 3. Graham JH & Menge JA, Phytophthora-induced diseases. Packer L., Vol. 105. Academic Press, Orlando, FL, USA) In: Compendium of Citrus Diseases, (Ed. LW Timmer, (1984) 121. SM Garnsey & JH Graham APS Press, St. Paul, MN), 20. Thomas RL, Jen JJ & Morr CV, Changes in solubleand (2000) 12. bound peroxidase – IAA oxidase during tomato fruit 4. Timmer L, Zitko SE, Gottwald TR & Graham JH, development. J Food Sci, 47 (1981) 158. Phytophthora brown rot of citrus: Temperature and 21. Zhu Q, Maherfl EA, Sameer Masoud RA & Lamb CJ, moisture effects on infection, sporangium production and Enhanced protection against fungal attack by constitutive dispersal. Plant Dis, 84 (2000) 157. co-expression of chitinase and glucanase in transgenic 5. Kaur A, Verma KS & Thind SK, Screening of different tobacco. Biotechnology, 12 (1994) 807. citrus rootstocks against foot rot disease (P. nicotianae var. 22. Bradford MM, Rapid and sensitive method for the parasitica). Plant Dis Res, 28 (2013) 49. quantitation of microgram quantities of protein utilizing 6. Das AK, Fungal disease in citrus and its management: the principle of protein-dye binding. Anal Biochem, Integrated management in citrus. (National Research 72 (1976) 248. Center for Citrus Bulletin, Nagpur), (2009) 53. 23. Bremner JM. Inorganic forms of nitrogen. Methods of Soil 7. Graham JH & Timmer LW, Florida Citrus pest Analysis. Part 2. Chemical and Microbiological Properties, management guide: Phytophthora foot rot and root rot. Madison, WI: Am Soc Agron, (1965) 1179. Retrieved 15.02.2013, from University of Florida IFAS 24. Jackson ML, Soil Chemical Analysis. Prentice Hall of India extension, (2008) http://edis.ifas.ufl.edu/cg009. Pvt. Ltd., New Delhi, (1973) 452. 8. Verniere C, Cohen S, Raffanel B, Dubois A, Venard P 25. Duncan LW, Graham JH & Timmer L, Seasonal patterns & Panabieres F, Variability in pathogenicity among associated with Tylenchulus semipenetrans and Phytophthora spp. isolated from citrus in Corsica. Phytophthora parasitica in the citrus rhizosphere. Phytopathology, 152 (2004) 476. Phytopathology, 83 (1993) 523. 9. Bairwa SK, Shrivastav AK, Kumar P, Meena RS, 26. Matheron ME, Wright GC & Porchas M, Resistance to Chanderbhan & Koli R, Developing strategies for Phytophthora citrophthora and P. parasitica and nursery integrated management of Phytophthora root rot and characteristics of several citrus rootstocks. Plant Dis, 82 gummosis in Kinnow mandarin (Citrus reticulata). The (1998) 1217. Bioscan, 9 (2015) 1327. 10. Sandler HA, Timmer LW, Graham JH & Zitko SE, Effect 27. Mohammed EG, Belmehdi I & Zemzami M, Citrus of fungicide applications on populations of Phytophthora rootstocks in morocco: present situation and future parasitica and on feeder root densities and fruit yields of prospects. Acta Hortic, 1065 (2015) 313. citrus trees. Plant Dis, 73 (1989) 902. 28. Cheema SS, Dhillon RS & Kapur SP, Phytophthora 11. Graham JH, Evaluation of tolerance of citrus rootstocks blight- A serious disease of citrus nursery. Prog Farm, to Phytophthora root rot in chlamydospore infested soil. 26 (1990) 16. Plant Dis, 74 (1990) 743. 29. Graham JH, Root regeneration and tolerance of citrus 12. Blaker NS & MacDonald JD, The role of salinity in the rootstocks to root rot caused by Phytophthora nicotianae. development of Phytophthora root rot of citrus. Phytopathology, 85 (1995) 111. Phytopathology, 76 (1986) 970. 30. Desikan R, Hancock J & Neill S, Reactive oxygen species 13. Siviero A, Furtado EL, Boava LP, Barbasso DV & as signaling molecules. In: Antioxidants and Reactive Machado MA, Evaluation of inoculation methods for Oxygen Species in Plants (Ed. Smirnoff N., Blackwell Pub. Phytophthora parasitica on citrus seedlings and young Ltd). (2005) 169. plants. Plant Soil, 241 (2002) 243. 31. Taylor NL & Millar AH, Oxidative stress and plant 14. Albrecht U & Bowman KD, Inducible proteins in citrus mitochondria. In: Methods in Molecular Biology, Vol. 372: rootstocks with different tolerance towards the root rot Mitochondria: Practical Protocols (Ed. Leister D & pathogen Pyhtophthora palmivora. J Phytopathol, 155 Herrmann JM) Humana Press Inc., Totowa, NJ). (2007). (2007) 606. 32. Fleischmann F, Gottlein A, Rodenkirchen H, Lutz C & 15. Rao, MV, Gopinadhan P & Douglas PO, Ultraviolet- B and Osswald W, Biomass, nutrient and pigment content of ozone-induced biochemical changes in antioxidant beech (Fagus sylvatica) saplings infected with enzymes of Arabidopsis thaliana. Plant Physiol, 110 Phytophthora citricola, P. cambivora, P. pseudosyringae (1996) 125. and P. undulata. Forest Pathol, 34 (2004) 79. 16. Teranishi Y, Tanaka A, Osumi M & Fukui S, Catalase 33. Binghua Liu, Mingjun Li, Liang Cheng, Dong Liang, activity of hydrocarbon utilizing candida yeast. Agr Biol Yangjun Zou & Fengwang Ma, Influence of rootstock Chem, 38 (1974) 1213. on antioxidant system in leaves and roots of young apple 258 INDIAN J EXP BIOL, APRIL 2019

trees in response to drought stress. Plant Growth Regul, 67 and molecular investigations of Fagus sylvatica seedlings (2012) 247. infected with Phytophthora citricola. Forest Pathol, 41 34. Shuncai Wang, Dong Liang, Chao Li, Yonglu Hao, (2011) 202. Fengwang Ma & Huairui Shu, Influence of drought stress 41. Dubey AK & Sharma RM, Effect of rootstocks on tree on the cellular ultrastructure and antioxidant system in growth, yield, quality and leaf mineral composition of leaves of drought-tolerant and drought-sensitive apple lemon (Citrus limon (L.) Burm.). Sci Hortic, 200 rootstocks. Plant Physiol Biochem, 51 (2012) 81. (2016) 131. 35. Apel K & Hirt H, Reactive oxygen species: metabolism, 42. Stolzy LH, Labanauska CK, Klotz LJ & Dewolfe TA, oxidative stress, and signal transduction. Annu Rev Plant Nutritional responses and root rot of Citrus limon and Biol, 55 (2004) 373. Citrus sinensis under high and low soil oxygen supplies in 36. Hernandez P, Zomeno L, Arino B & Blasco A, the presence and absence of Phytophthora spp. Soil Sci, Antioxidant, lipolytic and proteolytic enzyme activities in 119 (1975) 136. pork meat from different genotypes. Meat Sci, 66 (2004) 43. Feichtenberger E, Effect of systemic fungicide applications 525. on growth responses and fruit yields of sweet orange trees 37. Sharma RM, Dubey AK & Awasthi OP, Physiology of in Phytophthora infested soil. Proc Int Soc Citrus (Citrus paradisi Macf.). as affected by Nurserymen, 5 (1997) 267. rootstock. J Hort Sci Biotechnol, 90 (2015) 325. 44. Whiley AW & Pegg KG, Leaf mineral nutrient 38. Almansa MS, Del Rio LA, Alcaraz C F & Sevilla F, concentrations and yield in Phytophthora root rot affected Isozyme pattern of superoxide dismutase in different avocado trees treated with phosphate phosphorus varieties of citrus plants. Physiol Plant, 76 (1989) 563. compounds. South Africa Avocado Grower’s Assoc 39. Boava LP, Cristofani-Yaly M, Stuart, RM & Machado Yearbook, 10 (1987) 103. MA, Expression of defense-related genes in response to 45. Ajengui1 A, Bertolini E, Ligorio A, Chebil1 S, mechanical wounding and Phytophthora parasitica Ippolito A & Sanzani SM, Comparative transcriptome infection in Poncirus trifoliata and Citrus sunki. Physiol analysis of two citrus germplasms with contrasting Mol Plant Pathol, 76 (2011) 119. susceptibility to Phytophthora nicotianae provides 40. Portz RL, Fleischmann F, Koehl J, Fromm, J, Ernst, D, new insights into tolerance mechanisms. Plant Cell Rep, Pascholati, ST & Obwald WF, Histological, physiological 37 (2018) 483.