BMC Plant Biology

METHODOLOGY ARTICLE Open Access

An efficient method for zoospore production,infection and real-time quantification of

blight incidence was also conducted which indicated no significant difference in disease incidence, but incubation

Sharma et al. BMC Plant Biology (2015) 15:90 DOI 10.1186/s12870-015-0470-0Legumes Pathology, International Crop Research Institute for the Semi-AridTropics, Patancheru 502324, Telangana, Indiaperiod delayed under elevated CO2 as compared to ambient level.

Conclusion: The zoospore infection method for Phytophthora blight of pigeonpea will facilitate the small and largescale inoculation experiments and thus devise a platform for rapid and reliable screening against Phytophthora blightdisease of pigeonpea. qPCR allowed a reliable detection and quantification of P. cajani in samples with low pathogendensities. This can be useful in early warning systems prior to potential devastating outbreak of the disease.

Keywords: Phytophthora stem blight, Inoculation technique, Elevated CO2, qPCR

* Correspondence: [email protected] cajani causing Phytophthora blightdisease in pigeonpea under elevatedatmospheric CO2Mamta Sharma*, Raju Ghosh, Avijit Tarafdar and Rameshwar Telangre

Abstract

Background: Phytophthora blight caused by Phytophthora cajani is an emerging disease of pigeonpea (Cajanuscajan L.) affecting the crop irrespective of cropping system, cultivar grown and soil types. Current detection andidentification methods for Phytophthora species rely primarily on cultural and morphological characteristics, theassessment of which is time-consuming and not always suitable. Sensitive and reliable methods for isolation,identification, zoospore production and estimating infection severity are therefore desirable in case of Phytophthorablight of pigeonpea.

Results: In this study, protocols for isolation and identification of Phytophthora blight of pigeonpea were standardized.Also the method for zoospore production and in planta infection of P. cajani was developed. Quantification of fungalcolonization by P. cajani using real-time PCR was further standardized. Phytophthora species infecting pigeonpea wasidentified based on mycological characters such as growth pattern, mycelium structure and sporangial morphology ofthe isolates and confirmed through molecular characterization (sequence deposited in GenBank). For Phytophthoradisease development, zoospore suspension of 1 105 zoospores per ml was found optimum. Phytophthoraspecific real-time PCR assay was developed using specific primers based on internal transcribed spacer (ITS) 1and 2. Use of real-time PCR allowed the quantitative estimation of fungal biomass in plant tissues. Detectionsensitivities were within the range of 0.001 pg fungal DNA. A study to see the effect of elevated CO2 on Phytophthora 2015 Sharma et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.

Sharma et al. BMC Plant Biology (2015) 15:90 Page 2 of 12BackgroundPigeonpea (Cajanus cajan (L.) Millsp.) is one of the im-portant legume crops of rainfed agriculture in the semi-arid tropics. The Indian subcontinent, Eastern Africaand Central America, three main pigeonpea producingregions of the world cultivates pigeonpea either as a solecrop or intermixed. As pigeonpea contains high level ofprotein and some essential free amino acids like methio-nine, lycine and tryptophan, the importance of the pigeon-pea to world vegetarian population is very significant. InIndia, pigeonpea is second most important legume afterchickpea and alone contributes 72.5% of world culti-vated area with 62.5% of world production [1]. The accel-erated susceptibility of pigeonpea to diseases in Indiansubcontinent is one of the main roots of its deterioratingproductivity.Phytophthora blight (PB), caused by Phytophthora cajani

is an emerging disease in pigeonpea [2,3]. Information ontotal economic losses in India caused by PB is not avail-able, but the disease is of rising importance since last afew years and has the potential to cause 100% yieldlosses in field conditions under favourable environment.Occurrence and widespread distribution of PB has beenreported in areas especially when excessive rains fallwithin a short span of time and hot and humid weatherpersists during the crop season [2]. There is a lack of com-prehensive knowledge on available resistant genotypes toP. cajani [3].Identification of Phytophthora species by conventional

diagnostic tests abased on morphology, and growth onselective media is time consuming, laborious and takesconsiderable skill. In addition, taxonomic expertise is re-quired for correct identification within the closely re-lated species. Again the prospective for quantification ofbiomass of pathogen is limited. In this regard, the aim ofthe study was also to develop a rapid, highly specific andvery sensitive method for the potential quantification ofP. cajani. The polymerase chain reaction (PCR) has longbeen used to detect the pathogens and is a highly sensi-tive and relatively fast method that allows detecting spe-cific target DNA molecules in a complex mixture, offeringan alternative to microbiological conventional proceduresin fungal diagnostic. One of the most important factors inthe development of such molecular methods is the reli-ability of the primer set and the targeted DNA sequenceof interest organism [4]. Nuclear rDNA including thesmall and large subunits, 5.8S and the internal transcribedspacer (ITS) region, proved to be an ideal target for spe-cific PCR primers, as each sequences is variable at thefamily, genus or species level [5]. The ITS region has beenshown to be largely conserved within Phytophthora spp.but differ across species [6,7]. Most importantly, sequence

information is available in this region for nearly all knownspecies of Phytophthora [6]. Consequently, we designedP. cajani specific primers within the ITS region. Thecurrent technique has the further advantage of being ableto be performed as real-time PCR, visualized using anintercalating dye such as SYBR green. Real-time PCR al-lows products to be distinguished based not only size butalso on sequence, because melt temperatures will differfor same size but distinct products [8,9].In order to establish a successful method for in vitro in-

fection of P. cajani, a standardised protocol is needed toculture at its pathogenic state and to isolate the zoospores.However, knowledge about the conditions which governinfection by zoospores of P. cajani is yet unknown. Severalworkers have described inoculation techniques using my-celium [10,11], sporangium [12] and zoospores [13,14] forobtaining infection with Phytophthora spp.; howeverP. cajani being a putatively novel species, very limited in-formation about laboratory protocols are available. Fur-ther, Amin et al. [15] recognized that P. cajani resembledP. drechsleri but noted that P. cajani produced largersporangia and undifferentiated sporangiophores. Theyalso considered that the homothallic nature of P. cajanidifferentiated it from P. drechsleri, which is considered tobe heterothallic according to Savage et al. [16]. However,Kannaiyan et al. [17] expressed the opinion that PB causedby P. drechsleri var. cajani and P. cajani are probably thesame. Detailed descriptions for both these pathogens havebeen provided in Phytophthora Diseases Worldwide byErwin and Ribeiro [18]. In current study, attempts havebeen made to establish an in planta infection system ofP. cajani in pigeonpea.Host and pathogens are influenced by the interactive ef-

fects of multiple climatic factors. If any one of the factorsof disease triangle (host, pathogen and environment) is al-tered, changes in the progression of a disease epidemiccan occur [19]. High humidity, rising temperature, ele-vated CO2 and depletion of O3, all is events of imperativedeviations in atmospheric components. Changes in theseimportant climatic factors play a direct or/and indirectrole in changes of pathosystem and in disease expression[20]. Anthropogenic emissions are drastically increasingthe concentration of atmospheric CO2, as in 1750 atmos-pheric CO2 was 280 ppm has increased to 400 ppm in2013 and the projected concentration to be1250 ppm by2095 [21]. Elevated CO2 directly alters growth, develop-ment, metabolism and plant physiology which, in turn,has an impact on pathogen invasion and disease progress[22]. Elevated CO2 can also modify plantpathogen inter-actions primarily through changes in host plants [23].Till now it is not clear in the literature, whether the dis-

ease severity is enhanced or diminished by a higher CO2.The study of Thompson and Drake [24] showed that ele-vated CO2 reduced powdery mildew (Erysiphe graminis)

on wheat and the severity of rust (Puccinia sparganioidesEllis & Barth) on C3 sedge (Scirpus olneyi) Grey. On the

other side, higher temperature and increased CO2 concen-tration are also posing higher threat by increasing fungaldisease perception namely late blight of potato, rice blastand sheath blight. In this context, there is a need to studyeffect of elevated CO2 on Phytophthora blight of pigeon-pea, as in the last decade the disease dynamics of pigeon-pea has changed drastically in India.The overall objective of the study was to (i) standardize

protocol for isolation and infection of P. cajani, (ii) assessthe effect of elevated atmospheric concentration of CO2on PB development and (iii) quantify P. cajani during inplanta infection.

ResultsIsolation of PhytophthoraPigeonpea plants with typical symptoms of Phytophthorablight were sampled from the infected fields of multiplelocations of two different states of India during 2012-2013

Morphological identificationThe growth pattern, mycelium structure and sporangialmorphology of the individual isolates of Phytophthorawere examined under microscope for identification ofthe species. Pathogen formed distinct colonies in ros-aceous, stellate or cottony pattern irrespective of isolates.The mycelium of the isolates was coenocytic and branched.Some isolates formed hyphal swellings on V8 agar. Karyo-typing of the isolates showed single or pair of nuclei withinthe hyphae. Sporangium produced by different isolatesvaried in structure from broadly ovoid, obpyriform toelongate and non papilate. Based on morphological char-acters, the pathogen was identified as Phytophthora cajani(Figure 1a-f). Amphigynous anthredia were also observedand resembled to Phytophthora cajani as given in themanual of Phytophthora: Identifying species by morph-ology and DNA fingerprints [25].

s

S

R

B

R

B

B

B

B

R

S

B

B

B

B

Sharma et al. BMC Plant Biology (2015) 15:90 Page 3 of 12and 20132014. Blight symptoms included brown to darkbrown lesions distinctly different from healthy green por-tions on main stem, branches and petioles. These lesionssometimes elongate and cause girdle and cracks on thestems. Varying levels of PB incidence was recorded in sur-veyed locations (5.25-69.9%) irrespective of years. Therewas no effect of soil type on PB incidence (Table 1). Atotal of 14 isolates of Phytophthora (nine from 20122013and five from 20132014), one isolate from Varanasi-UttarPradesh, one isolate from each district of Mahbubnagarand Adilabad of Telangana State and rest 11 isolates fromdifferent experimental fields (BP5, BP09, BP14, BP15,DHF4, RL17 and RM1) of ICRISAT were obtained (Table 1).The pure culture of the isolates was maintained on 20%tomato extract agar media at 15 1C in dark condition.

Table 1 Passport information of Phytophthora cajani isolate

S. no* Isolates code Sample site and yearof collection

Location

1 ICPC 1 RL17-2013 Patancheru, Telangana

2 ICPC 2 BP09-2013 Patancheru, Telangana

3 ICPC 3 RM1-2013 Patancheru, Telangana



6 ICPC 6 Mahbubnagar-2013 Telangana

7 ICPC 7 Adilabad-2013 Telangana

8 ICPC 8 DHF4-2013 Patancheru, Telangana

9 ICPC 9 Varanasi-2013 Uttar Pradesh




13 ICPC 13 BP14-2014 Patancheru, Telangana14 ICPC 14 BP14-2014 Patancheru, Telangana B

*Serial number.Molecular characterizationTo confirm the P. cajani at molecular level, the ITS se-quence of 5.8S rDNA of the 14 isolates was amplified andsequenced. The sequences were purified and were used ina BLASTn against the Phytophthora database (http://www.phytophthoradb.org/). The BLAST result confirmed theidentity of the isolates as P. cajani. The sequences weredeposited under the GenBank (Table 1). The pair-wise nu-cleotide sequence identity matrix showed 99-100% se-quence identity among the isolates. The phylogeneticrelationship among the isolates and as well as with otherPhytophthora spp. was made based on nucleotide se-quences of ITS region. In phylogenetic tree, it was foundthat all the isolates of P. cajani were grouped together inthe same clade with the nearest relative P. sojae isolate.

used in the study

oil type PB incidence (%) Length of ITS sequence(nt) submitted

Accessionnumber

ed 43.75 504 KJ010534

lack 10.20 534 KJ010535

ed 5.25 728 KJ010536

lack 7.59 698 KJ010537

lack 12.50 518 KJ010538

lack 24.40 772 KJ622200

lack 69.90 570 KJ622201

ed 58.74 698 KJ622202

andy loam 48.40 467 KJ622203

lack 18.31 686 KJ622204

lack 13.04 506 KJ622205

lack 25.00 687 KJ622206

lack 5.77 749 KJ622207lack 14.29 822 KJ622208

Sharma et al. BMC Plant Biology (2015) 15:90 Page 4 of 12There was no phylogenetic discordance within the P.cajani isolates (Figure 2).

Zoospores production and in planta infectionPathogenicity of all the 14 isolates was tested individu-ally on the susceptible cultivar of pigeonpea ICP 7119.The pathogenicity of the isolates was tested on 50 seed-lings. The complete mortality of seedlings was recordedtwo days after the inoculation. Lesions were seen on thestem and whole seedlings collapsed within 67 daysafter inoculation. All the 14 isolates were found to bevirtually equal pathogenic to host ICP 7119 (data notshown). The P. cajani isolate ICPC 1 was taken to de-velop a method for zoospores production and in plantainfection as it had shown consistent susceptible reac-tion. We developed the reproducible method for zoo-spore production from P. cajani and the method ofinoculation for rapid screening for resistance to PB inpigeonpea genotype. The zoospores produced by P. cajaniisolate ICPC 1 were measured under haemocytometer andinoculum concentration of 1.5 105 zoospores per ml wasfound to be optimum for PB development. The symptomsstarted after 2 days of inoculation and 100% seedlingmortality was recorded at 34 days post inoculation.

Figure 1 Morphology of Phytophthora cajani isolate ICPC 1: (a) growth(d) non-papillate sporangia; (e) zoospores; (f) oospore.Un-inoculated plants did not show any disease symp-tom throughout the tests.

PB incidence under elevated CO2Incubation period, measured as the time in days betweeninoculation and disease symptoms was significantly de-layed under elevated CO2 as compared to ambient (p 75% humidity. After seven days, seedlings were in-oculated with P. cajani zoospore suspension (1.5 105

zoospores/mL) so that the roots were submerged up tocollar region in each well. Similar number of seedlings in-oculated with only sterilized water served as un-inoculatedcontrol. The experiment was designed in completely ran-domized design (CRD) manner and disease incidence wasrecorded everyday up to the complete mortality. The crit-ical differences (CD) value of disease incidence was calcu-lated at 1% level at 36 h, 48 h and 72 h.For quantification of P. cajani infection in planta,

seedlings were harvested 0 h (immediate after infection),2 h, 20 h, 27 h (30), 48 h and 72 h of post inoculation,washed and preserved in 80C for further experiments.

Total genomic DNA (gDNA) extraction from plant samplesTotal gDNA from Phytophthora infected plant sampleswas isolated using PureLink Plant Total DNA Purificationafter the completion of incubation period and incubatedfor 20 h, in these cases with single changes of water. Fi-nally, mycelial growth of isolate was removed from theflask and zoospores were harvested in water suspension.The zoospores were checked under microscope and theconcentration of zoospores was measured in a haemo-cytometer. The suspension was used for in planta infec-tion with further dilution.

In planta infection system under elevated CO2The apparently healthy seeds of ICP 7119 were surfacesterilized in sodium hypochlorite (1% v/v) for 23 minwas evaluated in 0.8% agarose gel as well as by UV spec-trophotometry. The extracted DNA was stored at 20C.

Sharma et al. BMC Plant Biology (2015) 15:90 Page 11 of 12Development and evaluation of qPCR primersTo quantify the growth of P. cajani within the host tis-sue under different experimental conditions (E1, E2 andE3), the real-time PCR was carried out. Three pairs ofsequence specific primers were designed from internaltranscribed spacer (ITS) sequences of P. cajani usingIDT Primer Quest software (eu.idtdna.com/Primerquest/Home/Index). ITS sequences (Acc nos. KJ010534-KJ010538)were aligned using BioEdit v. 7.2.5. and primers were de-signed from conserved region of the ITS sequences. Totest the specificity of the primers pairs, PCR was carriedout separately with the individual primers sets using puregDNA of P. cajani in 50 L reaction mixture containing:5 L of 10 X Taq polymerase buffer, 1.5 L 50 mMMgCl2,1 L of 10 mM each dNTP, 1 M of each primer, 1 L of5u/1 L Taq polymerase (Invitrogen, USA), about 100 ngof gDNA and H2O up to 50 L. Reaction conditions were:94C for 4 min, (94C for 45 s, 60C for 45 s and 72Cfor 30 s) 35 cycles followed by incubation at 72C for10 min. The amplified products were separated by 1.5%(w/v) gel electrophoresis and sequenced. The expectedsize of PCR amplicons was constantly generated from thereal-time PCR analysis.

Real-time PCR reactionReal-time PCR was carried out in a total volume of20 L consisting of 10 L 2x SYBR Green PCR MasterMix (Applied Biosciences, USA), 500 nM of each primer(qPCR_F2 and qPCR_R2) (Table 2), and 1 ng of each tem-plate DNA. Sterile bi-distilled water was added up to afinal volume of 20 L. The PCR thermal cycling conditionswere as follows: 50C for 2 min, 95C for 10 min, 40 cyclesof 95C for 10 s, and 62C for 30 s (during which the fluor-escence was measured). Following the final amplificationcycle, a melting curve was constructed by measuring thefluorescence continuously when heating from 60 to 95Cat the rate of 0.5C per second. qPCR reactions were per-formed in a PikoReal 24 Real-Time PCR Detection System(Thermo Scientific, USA). To generate the standard curve,DNA from pure culture of P. cajani was subjected toqPCR with a 10-fold dilutions ranging from 10 ng to0.01 pg under the same conditions described above. Quan-tification values were automatically determined by thePikoReal software version 2.0 (Thermo Scientific, USA)and the threshold cycle (Ct) values were then obtained.The standard curve is a plot of the Ct versus log DNA con-centration. In all the experiments, appropriate negativecontrols containing no template were subjected to thesame procedure to eliminate or to detect whether any pos-sible DNA contamination present. Each sample was ampli-fied in triplicate replication in every experiment.AbbreviationITS: Internal transcribed spacer; qPCR: Quantitative polymerase chain reaction;PB: Phytophthora blight; rDNA: Ribosomal DNA; ICRISAT: International CropsResearch Institute for the Semi-Arid Tropics; PARP: Pimarcin ampicillin,rifampicin and pentachloronitrobenzine; DI: Disease incidence; gDNA: GenomicDNA; UV: Ultra violet; BLAST: Basic local alignment search tool; NCBI: NationalCenter for Biotechnology Information; MEGA6.06: Molecular evolutionarygenetics analysis version. 6.06; CRD: Completely randomized design; CD: Criticaldifferences; IDT: Integrated DNA technologies; Ct: Cycle threshold.

Competing interestsThe authors declare that they have no competing interests.

Authors contributionsMS conceived the study, analysed the results and drafted the manuscript. RGparticipated in the design of the study, analysed the results and helped inwriting the manuscript. AT and RT helped in conducting and analysing theexperiments. All authors read and approved the final version.

AcknowledgementsThe funding support from Department of Science and Technology-ClimateChange Division and National Food Security Mission (NFSM), Department ofAgriculture & Cooperation, Ministry of Agriculture, Govt. of India is gratefullyacknowledged. The authors are thankful for technical assistance from Mr. BalKrishna from Legumes Pathology group.

Received: 4 December 2014 Accepted: 6 March 2015

References1. FAO. FAO Year book (Production). Food and Agriculture Organization of the

United Nations. 2012.2. Pande S, Sharma M, Mangla UN, Ghosh R, Sundaresan G. Phytophthora

blight of Pigeonpea [Cajanus cajan (L.) Millsp.]: an updating review ofbiology, pathogenicity and disease management. Crop Prot. 2011;30:9517.

3. Sharma M, Pande S, Rao JN, Kumar PA, Reddy DM, Benagi VI, et al.Prevalence of phytophthora blight of pigeonpea in the Deccan Plateau ofIndia. Plant Pathol J. 2006;22:30913.

4. Grnwald NJ, Martin FN, Larsen MM, Sullivan CM, Press CM, Coffey MD,et al. Phytophthora-ID.org: a sequence-based Phytophthora identificationtool. Plant Dis. 2011;95:33742.

5. Cooke DEL, Duncan JM. Phylogenetic analysis of Phytophthora speciesbased on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat.Mycol Res. 1997;6:66777.

6. Cooke DEL, Schena L, Cacciola SO. Tools to detect, identity and monitorPhytophthora species in natural ecosystem. J Plant Pathol. 2007;89:1328.

7. Lee SB, Taylor JW. Phylogeny of five fungus-like Protoctistan Phytophthoraspecies, inferred from the Internal Transcribed Spacer of Ribosomal DNA.Mol Biol Evol. 1992;9:63653.

8. Ririe KM, Rusmussen RP, Wittwer CT. Product differentiation by analysis ofDNA melting curves during the polymerase chain reaction. Anal Biochem.1997;245:15460.

9. Tajima K, Aminov RI, Nagamine T, Matsui H, Nakamura H, Bennoy Y.Diet-dependent shifts in bacterial population in the rumen revealedwith real-time PCR. Appl Environ Microbiol. 2001;7:276674.

10. Stewart S, Robertson AE. A modified method to screen for partial resistanceto Phytophthora sojae in soybean. Crop Sci. 2012;52:11816.

11. Tedford EC, Miller TL, Nielsen MT. A detached-leaf technique for detectingresistance to Phytophthora parasitica var. nicotianae in tobacco. Plant Dis.1990;74:3136.

12. Nowakowska M, Nowicki M, Kosinska U, Maciorowski R, Kozik EU. Appraisalof artificial screening techniques of tomato to accurately reflect fieldperformance of the late blight resistance. Plos One. 2014;9:112. doi:10.1371/journal.pone.0109328.

13. Ariadna M, Bosland PW. A rapid technique for multiple-race disease screeningof phytophthora foliar blight on single Capsicum annuum L. plants. Hort Sci.2010;45:15636.

14. Bouwmeester K, Govers F. A novel method for efficient and abundantproduction of Phytophthora brassicae zoospores on Brussels sprout leaf

discs. BMC Plant Biol. 2009;9:111.

15. Amin KS, Baldev B, Williams FJ. Phytophthora cajani, a new species causingstem blight on Cajanus cajan. Mycologia. 1978;70:1716.

Sharma et al. BMC Plant Biology (2015) 15:90 Page 12 of 1216. Savage EJ, Clayton CW, Hunter JH, Brenneman JA, Laviola C, Gallegly ME.Homothallism, heterothallism and interspecific hybridization in the genusPhytophthora. Phytopathology. 1968;58:100421.

17. Kannaiyan J, Ribeiro OK, Erwin DC, Nene YL. Phytophthora blight ofpigeonpea in India. Mycologia. 1980;72:16981.

18. Erwin DC, Ribeiro OK. Phytophthora diseases worldwide. St Paul, Minnesota,USA: APS Press; 2005.

19. Rogers DJ, Randolph SE. Climate change and vector-borne diseases. AdvParasit. 2006;62:34581.

20. Drake J. Population effects of increased climatic variation. Proc Royal SocLondon Series B Biol Sci. 2005;272:18237.

21. Savary S, Ficke A, Aubertot JN, Hollier C. Crop losses due to diseases andtheir implications for global food production losses and food security. FoodSec. 2012;4:51937.

22. Ainsworth EA, Davey PA, Bernacchi CJ, Dermody OC, Heaton EA, Moore DJ,et al. A meta-analysis of elevated [CO2] effects on soybean (Glycine max)physiology, growth and yield. Glob Change Biol. 2002;8:695709.

23. Chakraborty S, Luck J, Hollaway G. Impacts of global change on diseases ofagricultural crops and forest trees. CAB Reviews: Perspectives in Agriculture,Veterinary Science, Nutrition and Natural Resources 2008, 3 No. 054.

24. Thompson GB, Drake BG. Insects and fungi on C3 sedge and a C4 grassexposed to elevated atmospheric CO2 concentrations in open-top chambersin the field. Plant Cell Environ. 1994;17:11617.

25. Gallegly ME, Hong CX. Phytophthora: identifying species with morphologyand DNA fingerprints. St Paul, Minnesota, USA: APS Press; 2008.

26. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, et al.Nuclear ribosomal internal transcribed spacer (ITS) region as a universalDNA barcode marker for fungi. In Proceedings of National Academy ofScience: 2012; USA; 2012:624146.

27. Flier WG, Grnwald NJ, Kroon LPNM, Van Den Bosch TBM, Garay-Serrano E,Lozoya-Saldaa H, et al. Phytophthora ipomoeae sp. nov., a new homothallicspecies causing leaf blight on Ipomoea longipedunculata in the Toluca Valleyof central Mexico. Mycol Res. 2002;106:84856.

28. Cooke DEL, Drenth A, Duncan JM, Wagels G, Brasler CM. A molecularphylogeny of Phytophthora and related oomycetes. Fungal Genet Biol.2000;30:1732.

29. Hosseini S, Karlsson M, Jensen DF, Heyman F. Quantification of Phytophthorapisi DNA and RNA transcripts during in planta infection of pea. Eur J PlantPathol. 2012;132:45568.

30. Eye LL, Sneh B, Lockwood JL. Inoculation of soybean seedlings withzoospores of Phytophthora megasperma var sojae for pathogenicity andrace determination. Phytopathology. 1978;68:176973.

31. Chang SH, Kwack MS, Kim YS, Lee JY, Kim KD. A rapid radicle assay forpre-screening antagonistic bacteria against Phytophthora capsici on pepper.Mycobiology. 2001;29:21823.

32. Heid CA, Stevens J, Livak KJ, Williams PM. Real-time quantitative PCR. GenomeRes. 1996;6:98694.

33. Babu KB, Saxena AK, Srivastava AK, Arora DK. Identification and detection ofMacrophomina phaseolina by using species-specific primers and probe.Mycologia. 2007;99:797803.

34. Nair S, Roshna OM, Soumya VP, Hegde V, Kumar MS, Manimekalai R, et al.Real-time PCR technique for detection of areca nut yellow leaf diseasephytoplasma. Australas Plant Path. 2014. doi 10.1007/s13313-014-0278-7.

35. Nicolaisen M, Supronien S, Nielsen LK, Lazzaro I, Spliid NH, Justesen AF.Real-time PCR for quantification of eleven individual Fusarium species incereals. J Microbiol Meth. 2009;76:23440.

36. Jwa NS, Walling LL. Influence of elevated CO2 concentration on diseasedevelopment in tomato. New Phytol. 2001;149:50918.

37. Gautam HR, Bhardwaj ML, Kumar R. Climate change and its impact on plantdiseases. Curr Sci India. 2013;105:25.

38. McElrone AJ, Reid CD, Hoye KA, Hart E, Jackson RB. Elevated CO2 reducesdisease incidence and severity of a red maple fungal pathogen viachanges in host physiology and leaf chemistry. Glob Change Biol.2005;11:182836.

39. Madhu M, Hatfield JL. Dynamics of plant root growth under increasedatmospheric carbon dioxide. Agron J. 2013;105:65769.

40. Rouhier H, Read DJ. Plant and fungal responses to elevated atmosphericCO2 in mycorrhizal seedlings of Betula pendula. Environ Exp Bot.

1999;42:23141.

41. Nguyen C. Rhizodeposition of organic C by plant: mechanisms and controls.Sustain Agric 2009, 97123. doi: 10.1007/978-90-481-2666-8_9.Submit your next manuscript to BioMed Centraland take full advantage of:

Convenient online submission

Thorough peer review

No space constraints or color gure charges

Immediate publication on acceptance

Inclusion in PubMed, CAS, Scopus and Google Scholar

Research which is freely available for redistribution42. Evangelisti E, Govetto B, Minet-Kebdani N, Kuhn ML, Attard A, Ponchet M,et al. The Phytophthora parasitica RXLR effector penetration-specific effector1 favours Arabidopsis thaliana infection by interfering with auxin physiology.New Phytol. 2013;199:47689.

43. Reddy MV, Nene YL, Raju TN, Sheila VK, Sarkar N, Remanandan P, et al.Pigeonpea lines field-resistant to phytophthora blight. Int PigeonpeaNewslett. 1991;13:202.

44. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing offungal ribosomal RNA genes for phylogenetics. PCR Protoc: Guide MethodsAppl. 1990;18:31522.

45. Tamura K, Nei M. Estimation of the number of nucleotide substitutions inthe control region of mitochondrial DNA in humans and chimpanzees. MolBiol Evol. 1993;10:51226.Submit your manuscript at www.biomedcentral.com/submit

AbstractBackgroundResultsConclusion

BackgroundResultsIsolation of PhytophthoraMorphological identificationMolecular characterizationZoospores production and in planta infectionPB incidence under elevated CO2Development of qPCR for the quantification of Phytophthora cajaniSpecificity of the primersThe standard curvePhytophthora cajani colonization under elevated CO2

DiscussionConclusionsMethodsPlant materialFungal isolates and disease incidenceMorphological identificationIdentification with ITS sequencingPhylogenetic analysisStandardization of zoospore productionIn planta infection system under elevated CO2Total genomic DNA (gDNA) extraction from plant samplesDevelopment and evaluation of qPCR primersReal-time PCR reactionAbbreviation

Competing interestsAuthors contributionsAcknowledgementsReferences

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