Indian Journal of Experimental Biology Vol. 57, April 2019, pp. 248-258 Phytophthora nicotianae Breda de Haan induced stress changes in citrus 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 citrus rootstock genotypes including sour orange, Troyer citrange and six variants of C. jambhiri Lush. viz., RLC-5, RLC-6, RLC-7, Grambiri, rough lemon and Italian rough lemon 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, Rangpur lime, 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 Kinnow 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 citrus production. 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).