Potassium sulphate induces resistance of rice against the rootknot nematode Meloidogyne graminicola

LIU Mao-yan ,PENG De-liang ,SU Wen ,XIANG Chao ,JIAN Jin-zhuoZHAO JiePENG HuanLIU Shi-mingKONG Ling-anDAI Liang-yingHUANG Wen-kunLIU Jing

1 Key Laboratory for Biology and Control of Plant Diseases and Insect Pests,College of Plant Protection,Hunan Agricultural University,Changsha 410128,P.R.China

2 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

3 School of Agricultural Science,Xichang University,Xichang 615013,P.R.China

Abstract Potassium (K),an important nutrient element,can improve the stress resistance/tolerance of crops. The application of K in resisting plant-parasitic nematodes shows that the K treatment can reduce the occurrence of nematode diseases and increase crop yield. However,data on K2SO4 induced rice resistance against the root-knot nematode Meloidogyne graminicola are still lacking. In this work,K2SO4 treatment reduced galls and nematodes in rice plants and delayed the development of nematodes. Rather than affecting the attractiveness of roots to nematodes and the morphological phenotype of giant cells at feeding sites,such an effect is achieved by rapidly priming hydrogen peroxide (H2O2)accumulation and increasing callose deposition. Meanwhile,galls and nematodes in rice roots were more in the potassium channel OsAKT1 and transporter OsHAK5 gene-deficient plants than in wild-type,while the K2SO4-induced resistance showed weaker in the defective plants. In addition,during the process of nematode infection,the expression of jasmonic acid (JA)/ethylene (ET)/brassinolide (BR) signaling pathway-related genes and pathogenesis-related (PR)genes OsPR1a/OsPR1b was up-regulated in rice after K2SO4 treatment. In conclusion,K2SO4 induced rice resistance against M.graminicola. The mechanism of inducing resistance was to prime the basal defense and required the participation of the K+ channel and transporter in rice. These laid a foundation for further study on the mechanism of rice defense against nematodes and the rational use of potassium fertilizer on improving rice resistance against nematodes in the field.

Keywords: rice,Meloidogyne graminicola,potassium sulphate,induced resistance,H2O2,callose,potassium channel and transporter

1.Introduction

Rice is one of the most widely grown cereal crops in the world. The root-knot nematode (RKN)Meloidogyne graminicola(MG) is an important parasitic nematode in rice production,which roughly occurs in Asia,Africa,and other regions (Pokharelet al.2007;Mantelinet al.2016). Chemical control is the primary means to control this nematode but causes pollution to the environment.Instead,the environmentally friendly biological nematicides are sensitive to climatic,seasonal,and geographical conditions,thus affecting the efficacy of field control. Zhanet al.(2018a) identified only three cultivars with resistance against RKNs among 136 commercial rice cultivars in China. Induced resistance (IR) has been shown to reduce the harm of RKNs and ensure the safety of rice production (Huanget al.2015;Zhanet al.2018b).

Under the stimulation of elicitors (biotic or abiotic agents),the IR of plants was activated to avoid or reduce the harm of pathogens. The resulting resistance is usually broad-spectrum and persistent,so it is widely used to control plant diseases (Walterset al.2013).Spraying boron,zinc,and manganese on wheat leaves can improve rust resistance and greatly increase the enzymatic activities (i.e.,peroxidase and poly phenoloxidase) (Tohamey and Ebrahim 2015). Silicon treatment in roots can reduce the sensitivity of rice to MG,stimulate the basal defense response of the host,and increase the expression of defense-related genes of the ET pathway (Zhanet al.2018b). In addition,Soareset al.(2021) found that foliar spraying of silicate clay can also enhance rice resistance against MG. The treatment of potassium nitrate (KNO3) in roots can activate resistancerelated enzymes,the production of disease-resistant substances in tomatoes (SolanumlycopersicumL.),and then significantly reduce the RKNMeloidogyneincognitadisease index (Zhaoet al.2016). Xianget al.(2018)demonstrated that potassium fertilizer in the soil could stimulate the secretion of phenic acid (cinnamic,ferulic,and salicylic acids) and enhance the expression level of disease-resistant genes in plant roots,which effectively controlled soybean cyst nematodeHeteroderaglycines.Application of K2SO4in the field can reduce potato losses due to brown rot disease and up-regulate the expression of defense-related genes in snap bean leaf tissue(Mahmoud 2007;El-Garhyet al.2016). However,the data on plant nematode resistance induced by K2SO4is lacking.

In this work,the effect of K2SO4on the rice MG resistance was evaluated by pot experiment in a greenhouse,and the histopathological changes of rice treated with K2SO4were detected by microscopy. After the effect was confirmed,its role in priming the basal defense reaction was determined. Then,the expression levels of resistance-related genes were quantified to preliminarily explore the molecular mechanism. Finally,the lines deficient in potassium (K+) channel geneOsAKT1and transporter geneOsHAK5were used to verify whether the OsAKT1 and OsHAK5 proteins are involved in the host resistance.

2.Materials and methods

2.1.Plant materials and K2SO4

Wild-type (WT) rice seeds (Oryzasativacv.Nipponbare)were derived from the United States Department of Agriculture (GSOR-100) and cultivated in Changsha City,Hunan Province,China. TransgenicOsAKT1-RNAi lines deficient in K+channel OsAKT1 originated from Nipponbare (NPB) were generated using theAgrobacterium-mediated method described by Wanget al.(2016) and identified by qRT-PCR (Appendix A).TransgenicOsHAK5-Cr lines deficient in K+transporter OsHAK5 were generated from NPB by the CRISPR/Cas9(Sander and Joung 2014) and identified by PCR (Appendix B). In our experiments,rice seeds were soaked in 4-5%sodium hypochlorite (NaOCl) for 5-8 min for disinfection,then washed with distilled water three times,and then germinated at 28°C in the incubator. Budding seeds were planted in each polyvinyl chloride (PVC) tube containing Super Absorbent Polymer (SAP) substrates (1:400(w/v) mixture of sand and SAP) (Reversatet al.1999).Rice seedlings were maintained in a greenhouse at 28°C with 75% relative humidity and 16 h/8 h light/dark photoperiod and irrigated with 10 mL of K2SO4-free nutrient solution (NH4NO3,914 g;NaH2PO4·2H2O,403 g;CaCl2,886 g;MnCl2·4H2O,15.0 g;(NH4)6Mo7O24·4H2O,0.74 g;H3BO3,9.34 g;FeCl3·6H2O,77.0 g;and C6H8O7·H2O,119 g in 10 L water) every 3 days according to the manual of Cocket al.(1976). K2SO4(CAS 7778-80-5,purity not less than 99%,pH 5-8 at 25°C) was provided by Nanjing Chemical Reagent Co.,Ltd.,China.At first,K2SO4stock solution (400 mmol L-1) was prepared and then diluted at various concentrations (0.125,0.5,2,and 8 mmol L-1).

2.2.Collection,culture and hatching of MG

MGS collected from Pingjiang County,Yueyang City,Hunan Province,were identified with morphological and molecular methods according to Golden and Birchfield(1965) and Htayet al.(2016),respectively,and then subcultured on Nipponbare in a glass greenhouse at about 28°C. After the nematode was fully developed and mature,eggs were collected from root galls and incubated at 28°C for 72 h. The hatched second-stage juveniles(J2s) were filtered with a 25-μm sieve and diluted with distilled water into a suspension of ≈100 nematodes per mL for inoculation (Nusbaum and Barker 1966).

2.3.Direct effects of K2SO4 on MG

About 100 J2s were placed in a culture plate (φ 3.5 cm)containing 1 mL of diluted K2SO4solution (0.125,0.5,2,and 8 mmol L-1) or distilled water. After culturing for 72 h,a few drops of NaOH solution (1 mol L-1) were into the well of the plate. Then the mortality of nematodes was calculated under the stereomicroscope. Nematodes that responded to NaOH were judged alive,and the others were judged dead (Chen and Dickson 2000). The experiment was carried out thrice with four biological replicates each time.

About 100 newly hatched J2s were transferred in K2SO4solution (0.5 mmol L-1) or distilled water for 48 h and then inoculated on the 2-week-old rice planted in PVC tubes containing SAP substrates,then maintained in a greenhouse at 28°C with 75% relative humidity. At 14 days post-inoculation (dpi),the root samples were carefully cleaned,wrapped with microcloth,bleached with 5% NaOCl for 5-10 min,then rinsed with distilled water at least three times to remove residual NaOCl,and then dyed in acid fuchsin staining (Naharet al.2011). After the dye on the root surface was washed with distilled water,the roots samples were placed in glycerin to count nematodes in the rice roots at each developmental stage under the stereomicroscope. Galls of each treatment were randomly selected to take photos with Dual-Sensor Monochrome and Color Camera(Olympus DP80) under the microscope BX53 research microscope (Olympus Optical Company,Tokyo,Japan)at four magnifications. The proportion of nematodes of different ages was determined using Microsoft Excel 6.0(Redmond,Washington,USA). The experiment was performed thrice with six plants per replicate in each treatment.

2.4.Evaluation of rice resistance against MG induced by K2SO4

Two-week-old rice planted in the greenhouse was irrigated with 10 mL K2SO4dilution (0.125,0.5,2,and 8 mmol L-1) or distilled water one day before inoculation(≈100 J2s per plant). After inoculation,plants were continuously irrigated with K2SO4dilution or water twice a week. The cultivation,treatment and inoculation of rice in the subsequent experiments all refer to this method. At 14 dpi,the height and fresh weight of the plant were measured. Then,after determining the number of galls on the roots,nematodes in each developmental stage and sex were counted according to the method in the previous Section 2.3. The proportion of nematodes of different ages and the male/female ratio were calculated using Microsoft Excel 6.0. The experiment was carried out thrice with six plants per replicate in each treatment.

2.5.The attraction of rice roots to MG and giant cells phenotype after K2SO4 treatment

A trapping experiment was used to observe the attraction of roots to nematodes according to the method described by Wanget al.(2009). First,11.5 g of Pluronic F-127 powder (sigma Aldrich,Brussels,Belgium) was added into 50 mL sterile water,stirred on ice,and placed in a refrigerator at 4°C to dissolve it completely. The roots of 2-week-old rice were soaked in K2SO4dilution (0.5 mmol L-1) or distilled water. After 24 h,the root tip of about 1 cm was cut and placed in the center of the culture hole (φ 3.5 cm)containing 1 mL Pluronic gel and approximately 100 J2s.After 6 h at room temperature,under microscope BX53 research microscope,nematodes within 5 mm from the root tip were counted and then photographed with Dual-Sensor Monochrome and Color Camera. Then the state of attraction of roots to nematodes was well defined.The experiment was carried out thrice with six biological replicates per replicate in each treatment.

Giant cell phenotypes were observed according to the method described by Yuanet al.(2008). The concentration of K2SO4was set as 0.5 mmol L-1. At 7 dpi,the root galls of rice were fixed in 1× PIPES buffer overnight,then dehydrated with ethanol diluents step by step,and then embedded according to instructions of the Technovit 7100 Kit. CryoStar NX50 Cryostat (Thermo Fisher Scientific,MA,USA) was used to cut gall tissue into 10-μm slices. Following staining with 0.5% toluidine blue for 5 min,the slice was under BX53 research microscope for observing giant cells. The experiment was repeated twice with 10 galls per replicate in each treatment.

2.6.Microscopic observation of callose deposition and quantification of H2O2

The concentration of K2SO4was set as 0.5 mmol L-1.Callose deposition was detected according to Milletet al.(2010). At 7 dpi,root galls were fixed overnight in ethanol acetic acid solution,then diluted with ethanol for dehydration,and then stained with 1% aniline blue.Callose deposition was examined under ultraviolet light using an Olympus IX83 Inverted Microscope (Olympus Optical Company,Tokyo,Japan) and quantified in Image J Software. The entire experiment was repeated thrice with 20 galls per replicate in each treatment.

Four treatments were applied to 2-week-old rice plants in the greenhouse,including K2SO4treatment alone,water or K2SO4treatment before inoculation,and water treatment alone as a control. Root samples collected at 8,24,and 72 h post-inoculation (hpi) were treated according to the method described by Jiet al.(2015),then the H2O2accumulation was detected by the trichloroacetic acid(TCA) method described by Velikovaet al.(2000). The entire experiment was repeated thrice with four biological replicates per replicate in each treatment.

2.7.Relative quantitative analysis of expression levels of resistance-related genes

Four treatment groups as described in Section 2.6 were set up. The concentration of K2SO4was set as 0.5 mmol L-1. Total RNA was extracted from root sample of six plants using RNeasy Plant Mini Kit (QIAGEN,Germany),and cDNA was synthesized using PrimeScript? RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa,Japan).The primer pairs for qRT-PCR analysis of resistance-related genes and internal reference genes are listed in Appendix C.All qRT-PCR analyses were performed using a 7500-Fast Real-Time PCR System (Thermo Fisher Technologies,Beijing,China) with two independent biological repeats and three technical replicates. The relative expression of genes was calculated by 2-ΔΔCtmethod (Livak and Schmittgen 2001).

2.8.Effects of OsAKT1 and OsHAK5 on resistance of rice to MG

The primer sequences are listed in Appendix C. Rice seeds were irrigated with water or K2SO4solution (0.5 mmol L-1) and then inoculated with ≈100 J2s 2 weeks later.

Inoculation ofOsAKT1-RNAi lines andOsHAK5-Cr transgenic lines was a way to determine whether OsAKT1 and OsHAK5 were involved in rice resistance against MG. K2SO4solution at a concentration of 0.5 mmol L-1or distilled water were irrigated on 2-week-old plants,then≈100 J2s were inoculated on rice roots 24 h later. The wild-type Nipponbare was set as a control. At 14 dpi,plant susceptibility was investigated using the previous method. The whole experiment was carried out thrice with six plants per replicate in each treatment.

2.9.Statistical analysis of data

The mean,standard errors,and statistical significance of the data were calculated by SPSS version 25 (IBM,Armonk,NY,USA). The statistical significance of the data was analyzed by ANOVA using Duncan’s new multiple range test ort-test.

3.Results

3.1.K2SO4 did not impact the mortality and infectivity of MG at low concentrations

The direct toxicity of K2SO4to MG was determined by calculating the mortality of nematodes at different concentrations of K2SO4solution 72 h after treatment.Although the mortality of nematodes increased along with the increase of K2SO4concentrations ranging from 0.125 to 8 mmol L-1,there was no significant difference compared to the control ((5.0±0.7)%) (Fig.1-A). These results indicated that K2SO4was not toxic to MG within 72 h at all concentrations in the experimental setting.

To determine whether K2SO4directly impacted the infection and development of MG,the newly hatched J2s were soaked in 0.5 mmol L-1K2SO4solution for 48 h before inoculation. At 14 dpi,there was no difference in the number of juveniles and adult females between the K2SO4-soaked and water-soaked groups,which indicated that there was no direct effect of K2SO4on the infection of MG. At the same time,most nematodes developed into adult females,and there was no significant difference in the number of both third-stage juveniles (J3s) and fourth-stage juveniles (J4s) (J3+J4) or the number of adult females between the two groups (Fig.1-B and C). These results indicated that K2SO4did not affect the development of MG.

3.2.K2SO4 induced rice resistance against MG

To evaluate the rice resistance against MG induced by K2SO4,the K2SO4solution at different concentrations were applied to rice roots. The results showed that compared to the water treatment,all K2SO4-treatments could reduce galls and nematodes in rice roots,and there was no significant difference among the K2SO4treatments at 14 dpi (Fig.2-A and B). Root galls and nematodes decreased by (57.2±4.4) and (59.2±6.6)%under 0.5 mmol L-1K2SO4treatment,respectively(Fig.2-A and B). In addition,the proportion of adult females in plants treated with K2SO4((70.9±5.6)%) was significantly lower than in untreated plants ((90.7±5.1)%),while the proportion of juveniles (J3+J4) ((27.0±6.3)%)was higher than control ((6.0±3.2)%) (Fig.2-C). These results indicated that the application of K2SO4at low concentrations could reduce the infection of nematodes and delay the development of nematodes. Therefore,K2SO4solution at the concentration of 0.5 mmol L-1was used in all the subsequent experiments. We also found that the male/female ratio of nematodes in rice roots treated with K2SO4was lower than in the control(Fig.2-D),indicating that K2SO4treatment inhibited the sex differentiation of nematodes in rice. Meanwhile,there was no significant difference in the fresh weight and height of rice shoots/roots between K2SO4and water treatment (Fig.2-E and F),suggesting that application of K2SO4had no toxic effect on rice growth. All these results indicated that K2SO4could induce rice resistance against MG.

3.3.K2SO4-induced rice resistance against MG was achieved by neither impacting the attractiveness of rice roots to nematodes nor inhibiting the developmental phenotype of the giant cell

To determine whether the attraction of rice root to nematodes was affected by K2SO4treatment,nematodes in the range of 5 mm around the root tip were counted. At 6 hpi,the number of nematodes attracted to the K2SO4-treated tip (26.2±3.1) was not different from water-treated(25.7±2.8) (Fig.3-A and D). The results indicated that K2SO4treatment did not affect the attraction of roots to MG.

To determine whether the development of giant cells was affected by K2SO4treatment,root galls were collected for histological observation at 7 dpi. The average cross-sectional area of giant cells in the K2SO4-treated roots was not significantly different from untreated (Fig.3-B and E). We also counted giant cells in feeding sites;there was no significant difference between the two treatments (Fig.3-C and E). All these results indicated that the application of K2SO4did not influence the development of giant cells.

3.4.K2SO4 induced rice resistance against MG by priming basal defense

By observing the presence of callose in the gall tissue,we found that the K2SO4-treated group had more callose deposits and bigger and denser callose spots than the water-treated group (Fig.4-A and C),with a 67.9%increase in the average area of callose spots (Fig.4-A).In addition,compared to water-treated,the expression of the callose biosynthesis geneOsGSL1was up-regulated in the K2SO4-treated rice roots at 72 hpi,while the callose degradation geneOsGNS5was down-regulated(Fig.4-B). These results suggested that K2SO4treatment increased the deposition of callose in root galls during the rice defense against nematode infection.

The detection of H2O2content in rice roots revealed that the H2O2response could also be induced by K2SO4alone,as its H2O2level at 24 hpi was significantly higher than the control (Fig.4-D). In the nematode challenge,the level of H2O2in K2SO4-treated plants increased by 78.2 and 118.7% at 8 and 24 hpi,respectively,compared to the untreated plants (Fig.4-D). In addition,the expression ofOsRbohB,an H2O2biosynthesis gene known to be involved in plant immune response (Wong 2007),was up-regulated by K2SO4treatment in rice roots at 8 and 24 hpi (Fig.4-E). Especially at 8 hpi,the expression level ofOsRbohBwas significantly higher in inoculated plants treated with K2SO4than untreated plants(P＜0.05) (Fig.4-E),which was consistent with the sure H2O2content (Fig.4-D). These results suggested that K2SO4could induce rice defense against MG by activating the rapid production of reactive oxygen species (ROS).

3.5.K2SO4 could up-regulate the expression of some rice resistance-related genes

Firstly,two salicylic acid (SA) pathway-related genes,SA transcription factorOsWRKY45and biosynthesis geneOsICS1were analyzed. The expression of bothOsWRKY45andOsICS1was significantly increased by K2SO4treatment alone at 72 hpi. However,K2SO4treatment only enhanced the expression ofOsWRKY45at 24 hpi in infected plants (Fig.5-A).Therefore,K2SO4could direct activate defense depending on the SA pathway. Secondly,we analyzed two jasmonic acid (JA)pathway-related genes and two ethylene (ET) pathway-related genes. After treatment with K2SO4,the expression of the JA transcription factor geneOsJAMYBwas significantly increased(P＜0.05) in both inoculated and non-inoculated groups at 8 hpi(Fig.5-B). The expression pattern of JA biosynthesis geneOsAOS2was similar to that ofOsJAMYB,but it was up-regulated more thanOsJAMYBat 8 hpi (Fig.5-B).Although the ET signaling geneOsEIN2was inactive,its expression level was significantly higher in inoculated plants treated with K2SO4than untreated plants after 24 hpi. At the same time,the expression of ET biosynthesis geneOsACS1was increased sharply in inoculated plants treated with K2SO4,which enhanced 6.98-fold more than untreated plants at 72 hpi (Fig.5-C).These results suggested that K2SO4played an active role in inducing the expressions of JA and ET pathways-related genes during rice resistance to RKNs infection. Thirdly,we analyzed the expression of pathogenesisr elated (P R) genes.The expression of bothOsPR1aandOsPR1bsignificantly increased in K2SO4-treated plants compared to untreated plants at 8 hpi.Moreover,the expression level ofOsPR1bcontinued to increase after 8 hpi in infected plants treated with K2SO4and remained significantly higher than untreated plants at 24 hpi (Fig.5-D). These results suggested that K2SO4positively regulated the expression of these two PR genes during rice resistance against RKNs.

Finally,the expression of two brassinolide (BR)pathway-related genes was analyzed. The expression of BR receptor geneOsBRI1was significantly increased in both infected and uninfected plants treated with K2SO4at 24 and 72 hpi,while the expression level of BR biosynthesis geneOsDwarfwas significantly higher in infected plants treated with K2SO4than untreated plants at 24 hpi (Fig.5-E).This result indicated that the BR pathway could play an active role during the resistance process,and K2SO4positively regulated the expression of BR pathway-related genes.

3.6.The K2SO4-induced rice resistance against MG required the participation of the potassium absorption and transportation system

To determine the role of K+absorption and transportation systems in the resistance process,we first compared the expression of K+channel geneOsAKT1and K+transporter geneOsHAK5between K2SO4treatment and non-treatment,and nematode inoculation and non-inoculation. The expression level ofOsAKT1was higher in the inoculated plants treated with K2SO4than in others within 72 hpi,especially at 8 and 72 hpi(Fig.6-A). These suggested thatOsAKT1expression was affected by K2SO4treatment and nematodes infection,which might be involved in rice resistance against nematodes induced by K2SO4.Meanwhile,the expression ofOsHAK5was up-regulated in each treatment within 24 hpi after inoculation. Although there was no significant difference among the treatments,the expression level ofOsHAK5was slightly higher in infected plants than in uninfected plants and K2SO4-treated plants than in untreated plants within 24 hpi (Fig.6-B). This indicated that the expression ofOsHAK5was not significantly affected by nematode infection and K2SO4treatment.

Subsequently,we inoculated nematodes on the rice lines deficient in these two genes.Interestingly,in the presence of K2SO4at a concentration of 0.5 mmol L-1,both of the two defective plants (OsAKT1-RNAi andOsHAK5-Cr) became more susceptible than the wild-type(Fig.6-C and D). Specifically,compared to wild-type,both galls and nematodes increased in these two defective plants,and the proportion of juveniles(J3+J4) reduced while the proportion of adult females increased (Fig.6-C-F). These results suggested that the deletion of these two genes leaded to reduced resistance to MG infection in rice. On the other hand,in the case of K+starvation,the susceptibility of these two defect types was still higher than that of the wildtype (Fig.6-C-F). This result suggested that these two genes could play an independent role in rice nematode resistance.However,K2S O4-induced resistance was weaker in plants with defects in these two genes than in the wild-type. (Fig.6-CF). This result indicated that K2SO4-induced rice resistance against MG required the participation of these two genes.

4.Discussion

4.1.The primary mechanism of K2SO4-induced rice resistance against MG was to prime the basal defense

Potassium can enhance plant resistance to biotic stresses(Holzmuelleret al.2007). The present study showed that K2SO4treatment could improve the resistance of rice and reduce the infection and development of nematodes,and had no direct effect on nematodes,which was similar to the treatment of silicon or biochar on nematodes host (Huanget al.2015;Zhanet al.2018b). The treatment of K2SO4at low concentrations could reduce the susceptibility of rice to MG,and at higher concentrations was not conducive to enhancing the control of nematodes,which was consistent with the control effect of K+onM.incognitaandH.glycines(Zhaoet al.2016;Xianget al.2018). The reproductive mode of MG is a combination of cross-fertilization and facultative meiotic parthenogenesis (Bellafioreet al.2015).Sexual reproduction is conducive to releasing higher genetic diversity (Kondrashov 1993). The depletion of homoglutathione content leads to an increased proportion of males (Baldacci-Crespet al.2012),and K2SO4deficiency treatment could lead to the similar results in our research,which was consistent with the view that sex determination of parthenogenetic RKN species is affected by environmental factors (Castagnone-Sereno 2006).

In nature,the attraction of the host roots to nematodes is of great importance in establishing infection (Aliet al.2011). Certain substances can affect the attraction of rice roots to MG,such asAspergilluswelwitschiae(Liuet al.2019;Xianget al.2020). Some substances do not have this effect,such as those that can induce rice resistance to nematodes infection (Silicon and thiamine,etc.) (Jiet al.2015;Huanget al.2016). K2SO4had no significant effect on the attraction of rice to nematodes,indicating that it might have the same effect. When the nematode moved and invaded the host plant,the infective J2s migrated to the vascular column cells then induced the root cells to differentiate into specialized giant cells for feeding sites to acquire the nutritional needs for subsequent growth and development (Bird 1996). Then the giant cells develop to form the root galls. Previous research observed that the morphology or size of giant cells could be affected by the treatments of β-aminobutyric acid (BABA) or αβdehydrocurvularin (Jiet al.2015;Xianget al.2020).However,K2SO4treatment had no significant effect on the phenotype of giant cells,which was consistent with the potassium treatment on tomato roots infected byMeloidogynejavanica(Yuanet al.2008).

H2O2is a signal molecule that mediates plant defense response to biotic or abiotic stress (Neillet al.2002). In most cases,the characteristic of host plant resistance is local necrosis of plant cells at the site of infection,also known as hypersensitive reaction (HR),which is by a burst of ROS (Williamson and Hussey 1996). In the induced resistance againstM.javanica,the production of H2O2may be helpful for tomato and cucumber plants to defend against RKNs infection (Sahebani and Hadavi 2009;Sahebaniet al.2011). Ascorbate oxidation activated systemic defense against MG,which was also related to the accumulation of H2O2(Singhet al.2020).Overexpression of respiratory burst oxidase homolog B (RbohB),a gene associated with H2O2generation,enhanced the resistance ofArabidopsisto nematodes(Hawamdaet al.2020). On the contrary,the interference or silencing of theRBOH1gene,which encodes NADPH oxidase for the production of apoplastic H2O2,increased the susceptibility of tomatoes toM.incognita(Songet al.2018). In our experiment,K2SO4treatment could upregulate the expression ofOsRbohBand enhance the accumulation of H2O2in infected rice roots within 24 hpi,indicating that K2SO4could induce rice resistance to MG infection by rapidly activating ROS bursts.

Callose deposition is one of the markers of plant defense response,which can play a role in resistance to pathogen infection (Mauch-Maniet al.2017). Ellingeret al.(2013) observed that spraying pathogen elicitor flg22 can induce callose deposition on leaf and increase the resistance ofArabidopsisto powdery mildew in the early stage. Bianet al.(2020) found that treatment with validamycin A significantly enhanced callose deposition inArabidopsisleaves to defend against different fungi. In the defense process of rice to MG,BABA can promote the deposition of callose at the rice root galls,increase the expression of callose biosynthesis geneOsGSL1,and reduce the expression of callose degradation geneOsGNS5(Jiet al.2015). In our study,the deposition of callose and the expression level ofOsGSL1andOsGNS5showed similar changes after K2SO4treatment,indicating that K2SO4might have a similar mechanism as BABA in priming the basal defense in the infected host.

4.2.K2SO4 treatment up-regulated the expression of some rice resistance-related genes in the process of rice resistance to MG infection

While the plant is attacked by the pathogens,it will activate the autoimmune response system to defense.The immune response is a complex process involving the transcription and expression of many resistancerelated genes which are mediated by different hormones(Panstrugaet al.2009;Naharet al.2011). The deletion of genes related to defense signaling pathways (e.g.,JA,ET,and BR) will result in the reduction of plant defense against pathogens (e.g.,MG,Botrytiscinerea,andM.incognita) (Naharet al.2011;Songet al.2018;Bianet al.2020). After the JA pathway was antagonized by gibberellin and monocrotaline,rice would be more susceptible to RKNs (Yimeret al.2018;Lahariet al.2019). The resistance of rice to MG induced by BABA or ascorbate oxidation depended on JA and ET pathways(Jiet al.2015;Singhet al.2020). The enhancement of rice resistance against MG induced by biochar or silicon was related to the increased transcription levels of ET pathway-related genes (Huanget al.2015;Zhanet al.2018b). Our results show that K2SO4treatment can also up-regulate the expression of these two genes during rice resistance to nematode infection. Moreover,the expression level of ET pathway-related genes increased after the JA pathway-related genes increased,which was consistent with the dependence of the ET pathway on JA biosynthesis during rice resistance to RKNs infection(Naharet al.2011). PR proteins are a vital component of plant defense system,and their accumulation at the site of infection leads to HR (Edreva 2005),usually accompanied by the accumulation of H2O2(Williamson and Hussey 1996).OsPR1aandOsPR1bcan be induced by exogenous plant hormones JA,SA,and ET,and play an important role in rice resistance (Agrawalet al.2001;Naharet al.2011). These are in line with our results and coincide with previous H2O2accumulation assays,indicating that K2SO4might increase the resistance of rice to nematodes through HR. BRs play an active role in plant innate immunity and enhance the level of H2O2in the apoplast (Nakashitaet al.2003;Xiaet al.2009).Naharet al.(2013) observed that a high concentration of exogenous epibrassinolide significantly reduced the susceptibility of rice to MG. In our study,while K2SO4treatment significantly increased the expression of BR pathway-related genes in rice roots,H2O2accumulation was also enhanced,which is similar to the result that BRs enhanced the accumulation of H2O2(Xiaet al.2009).However,further research is needed to elucidate the role of K2SO4-induced hormone metabolism in the process of rice resistance to MG infection.

4.3.The mechanism of K2SO4-induced resistance involved the K+ channel and transporter

Potassium plays a key role in maintaining the activity of cytoplasmic enzymes (Leigh and Wyn Jones1984). If potassium deficiency occurs,the normal physiological and biochemical reactions of plants will be correspondingly inhibited (El-Gendyet al.2015). However,most K+in the soil is dehydrated and cannot be used by plants (Maathuis 2009). Venkatesanet al.(2013) observed decreased K+content in rice after MG infection. In order to improve the utilization efficiency of K+,plants ensure the absorption and transport of K+invitroandinvivothrough K+transporters and channels (Wang and Wu 2013). Shiet al.(2018) observed that the effector protein AvrPiz-t fromMagnaportheoryzae,a pathogen of rice blast,targets the K+channel OsAKT1 to destroy plant immunity,and the deletion ofOsAKT1resulted in the decrease of K+content and the resistance againstM.oryzae. The high-affinity K+transporter HAK5 could interact with Raf-like Kinase ILK1,which is helpful to plant defense against bacterial pathogens and is necessary for innate immunity (Braueret al.2016). Our data showed that both OsAKT1 and OsHAK5 play independent and positive roles in the resistance of rice to MG and the K2SO4-induced resistance requires the participation of these two genes,which further confirms the previous views.Kyndtet al.(2017) found that subterranean infestation of root-knot nematodes predisposes rice to rice blast. The mechanism of decreased resistance of rice to MG caused by OsAKT1 deletion might be similar to that ofM.oryzaedestroying the immune system of rice (Shiet al.2018).The positive response to K2SO4treatment and nematode infection also showed thatOsAKT1might have a great initiative in regulating the loss of K+and maintaining the dynamic balance of K+,to reduce the internal competition of pathogens for nutritional resources,protect the activity of plant enzymes,and improve the synthesis of defense compounds (Holzmuelleret al.2007;Sarwar 2012).OsHAK5 is active in the mobilization of potassium in rice,and it not only mediates the absorption of K+in the external environment,but also mediates the transport of K+from root to shoot or from source to sink in plants(Yanget al.2014),but its expression is inhibited when the external K+is sufficient (Rubioet al.2014). Therefore,in our experiment,the expression level ofOsHAK5in K2SO4alone treatment was slightly lower than in others at 24 hpi.Anyway,the above results indicated that OsAKT1 and OsHAK5 contributed to the host’s resistance against MG and K+played a major role in this process.

5.Conclusion

This study further expanded the data on potassium in plant resistance to biotic stress by analyzing the function of potassium in rice resistance against MG. Treatment of K2SO4at low concentrations could improve the defense of rice against MG. K2SO4-induced resistance is achieved by initiating the basal defense of rice and with the participation of the K+channels and transporters.These results laid a foundation for further study on the mechanism of rice to defend against RKNs. However,the mechanisms of action of resistance-related genes and the K+channel and transporter need further exploration to better understand IR. In any case,K2SO4-induced resistance will provide theoretical guidance for the integrative control of nematodes in rice plants.

Acknowledgements

This work was supported by the Natural Science Foundation of China (32172382,31801716,and 31571986),the National Key Research and Development Program of China (2021YFC2600404),and the Scientific Research Project of Hunan Provincial Department of Education of China (19B259). The authors would like to thank Prof.Ning Yuese from Institute of Plant Protection,Chinese Academy of Agricultural Sciences,for providing theOsAKT1-RNAi lines seeds.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm