Screening for Ascochyta Blight Resistance in Chickpea Under Controlled Environment and Field Conditions M . P . H A W A R E , S e n i o r Scientist (Pathology), H . A . V A N R H E E N F , N , Principal Scientist (Breeding), a n d N . S . S . P R A S A D , S e n i o r Engineer, International C r o p s Research I n s t ~ t u t efor t h e Semi-Arid 'I'ropica ( I C R I S A T ) . P a t a n c h e r u , A n d h r a P r a d e s h 502 324, l n d i a ABSTRACT Haware. M P., van Rheenen. H. A,. and Prasad. N. S. S 1995. Screening for Ascochyta blight resistance In chlckpea under controlled environment and field cond~tions Plant I11r. 79:122-135. The occurrence and severity of Ascochyta blight of chickpea 1s influenced by env~ronrnenlal Pactors, which can complicate field screening. A control;ed-env~ronrnent plant growth room was developed at ICRISAT Asia Center to rapidly screen ch~ckpeagenotypes within 15 day, for resistance to Ascochyta blight caused by Ascochyra rahret. A posltlve correlation was obcerved between results from controlled-environment and field screenings In an area where the pathogen is endemic. Ascochyta blight, a fungal disease of chickpea (Cicer arierinum L.) caused by Ascochyra rabiei (Pass.) Lab., can devastate chickpea crops over large areas if weather conditions favor infection and spread of the pathogen. The years 1979-80 and 1981 -82, for instance, were disastrous for chickpea production in Submitted u ICRlSAT Journal Ankk 1649) Accepted 0 for publication 19 Onober 1994. 1995TheArnericanPhytopathologicalSoc1ety I32 Plant DiseaseNol. 79 No. 2 Pakistan and in the Punjab and Haryana states of India, where 40 50%) crop losses were reported (2). Breeding for resistance to Ascochyta bligl~thas been attempted in several countries ( 5 ) . Screening for Ascochyta blight resistance is usually carried out in field nurseries, using debris from diseased plants as inoculum, keeping the humidity high by providing irrigation at least twice aday, and screenine when weather conditions are naturally favorable to disease development (1,j). However, at places such as I C R I S A T Asia Center, Patancheru, lndia (18"N. 7g0E), where temperatures are relatively high and humidity low, Ascochyta blight - field 5creenlng i\ not poss~hlc,and the only option is to screen under controlled crlv~ronmentalcond~tlons.'T'hls method has advantages of uniformity, repeatahllity, Independence of the scason, and reduced risks of the disease spreading to chickpea crops. Ascochyta blight of chickpea IS influenced by environment. In controlled-environment studies, it was concluded that the b a s ~ crequirement for severe infection is a 17-hr wetness period in 2-wk-old seedlings at about 20 C (6). However, small temperature changes in near-saturated air will affect condensation on leaves. Therefore, to create a disease artificially, good control of temperature and humidity is very important for successful infection and disease development. This paper provides details of the construction and testing of a controlledenvironment screening facility. MATERIALS A N D METHODS Physical arrangement. The plant growth room is 9.75 m long X 6.32 m wide X 3 m high. Sixteen mild steel (MS) painted racks, each containing four shelves, were installed in the room (Fig. I). Each rack is 137 crn long X 122 cm w ~ d e ,and the space between shelves is 76 cm (Figs. I and 2). Temperature and humidity. An airconditioning unit (Model IOTR. 120.000 RTII. Westinghouse F.lectric. Pittsburgh. PA) was installed with suitable ducting arranged as shown in Figure I. Three separate waterproof switches were in~tallcd to control the air-conditioning system within the plant growth room. The plant growth room can be maintained between 15 C and 30 C. A humidifier ( Defepser-AG Type 13T. Welter Meier Holding AC, Switzerland) was mounted on a wall. 2 m above floor level. T h e unit functions between r o o m temperatures 5 C and 35 C and maintains constant relative humidity ( R H ) at 80-85%,, which allows continuous leaf wetness as long as required. A humidistat ( H R K - I ) supplied with the humidifier was installed on thc opposite wall of the chamber to the humidifier to regulate the humidity in the plant growth room. Lighting. On the top of each shelf, four 40-W fluorescent lights were provided, each fixed in a heavy-duty box made of 21 gauge sheet steel and painted to prevent rusting. In all, 256 fluorescent lamps were installed in these fix!ures. The ~lluminationwas further enhanced by using polished stainless steel reflectors (irradiance of 54 W m '). A timer was installed to automatically control the entire lighting system. Wiring. Each rack was supplied with a single phasc 230-V AC 50-H7 supply vla four 5A weatherproof switches so that lights on each shelf could be controlled individually. All of the 256chokes were mounted together in an adjoining room so that they did not affect the temperature and humidity in the plant growth room. The wires between each fluorescent light and its choke were laid through a central wiring PVC duct connecting all racks. All electrical installations in the plant growth room were grounded through a 2-mm bare coppcr wire according t o Indian Standard Specifications. Screening method. Twenty chickpea lines and an Ascochyta blight susceptible control P b 7 were grown in Ascochyta blight nurseries at Hisar in India. Each trial had at least two replicates. They wcrc sown in October- November 1990 and 1992. Another set of 10 chickpea lines received from Islamabad, Pakistan, was sowed at Hisar in November 1991. Plot size in the nurseries was in 4-m rows, with plant spacings of 30 crn between and 10 cm within rows. In the field, disease incidence was enhanced by spreadingdebris from Ascochyta blight infested chickpea plants between plant rows, and by spraying spore suspensions onto the plants. For the plant growth room studies, seeds were sowed in 80 plastic trays (35 X 25 X 8 cm) in sterilized river sand. There were 10 seedlings in each line in three replicates. Trays were transferred to the plant growth room approx~mately 2 wk after sowlng. A monoconidial isolate of .1, rohiri from a chickpea field at Hisar was used In the experiment,. The cultures were stored on potatudextrose agar at 5 C. The spores were produced on ch~ckpeaseed, prepared b! autoclaving 100 g of chickpea seed In 50 ml of water for 30 min in a flask. Thr seeds were inoculated from a 7-day-old culture of A . rahier and incubated lor 10 days at 20 C. The spore suspension was made by soaking infected seed In sterile distilled water for 30 min, stlrring with a glass rod, and passing the suspension through douhle-layered rtiuslin cloth. The suspensron was adjusted to the required spore concentration using Hum~d~fler Defenser-AG T~pe- 13T 200-240v, 50hZ, 1OOwatts --- - a hemacytometer. Seedlings were Inoculated after transfer to the plant growth room by spraying spore suspcnslonb ( 2 \ 10' spores per m ~ l l ~ l ~ tof e r )a tiisar sola ate of A , r a b i r ~ .Air temperature war m a ~ n t a ~ n eatd 20 i' (iI C') In the plant growth rooni. I.eaf uetness was niaintalned lor 72 hr using the hum~d~l'icr. Keliitive h u m l d ~ t y iri the rooni was r~ialnta~ned brtwr.cn 65 and 70"i during the \uhsequent I2 days. 111 the plant grouth rooni, the fluorescent I~ghtsprotldcd a 12-hr photoperiod. I)isease \cores were rccordcd ;it podJ ~ n gIn the field nurserit\ and ? wk alter inoculation 11) the plant growth rooni. Plants were scorcd on a scale ol I 9 ah docrlbed hq Nene ct ill (21, whcrc I no symptom\ and 9 pl;ints k~lled l'hc ; Blower Cod Unlt Make - Westlnghouse Model no - AH0 4OCAF - ~* 632 -i IT" C)(.J] r/ Llght Rack Duct Outlet Humidistat Sliding Door Tubelight * -476 -J Light Rack t 76 + 7- ' 15 Section - 66 ' - T 15 Section AA Fig. 1. Plant growth chamber at ICRISAT Asia Ccnlcr, Patanchcru, India. All dimens~ons are in centimeters. Plant DiseaseIFebruary 1995 133 repeatability of the plant growth room screening was investigated by testing chickpea lines at least twice. Correlation coefficients were calculated for t h e relationship between diseabe scores from the Ascochyta blight nursery at Hisar and the plant growth room at IC'RISAT Asia Center. scores were highly correlated. The I0 chickpea lines from Pakistan (Table 2) were resistant t o Ascochyta blight when f ~ e l dscreened at Islamabad in 1989-90 and 1990 91 ( 8 . A. Malik,personalcommunicarion). T h e higher disease scores in the plant growth room were apparently more d u e t o uniform and favorable temperatures and relative humidity. T h e disease reaction at the seedllng stage in t h e plant growth r o o m and disease assessment at podding in the field were RESlJLTS AND DISCllSSlON T h e results of screening materials in the plant growth room at I C R I S A T Asia Ccntcr and under field conditions at Hisar are given in'Tables I and 2. T h e Table I. Reactioii ot 21 chickpea l~ncsto Ascochyta hlight In the Ascochyta blight nursery (ABN) at HI^ and in the plant growth room at ICRISAT Asia Center, I'atancheru d u r ~ n r1990 91" Disease scoreb ('hickpea line ABN Plant growth ruom ICC' 607 ICC 1065 I('(' 1400 ICC 1472 I('C' I2967 I('C 13416 ICC' 138lO I('C 1491 1 ICC'I 86446 ICC'I. 86447 IC'CV $9445 ICCX 790151 I('CX X(H)X9 ICC'X XOOX59 I('CX XI0457 I W X 810737-1 IC('X $10737-2 ICCX X I0XOO ICC'X XI0974 I('CX 830677 Ph 7' "r: 0.9067: R' 0.82. hlI)isc;~re score I no symptoms to 9 = killed by the pathogen. 'Suscept~hlrcheck. 'Table 2. Reaction ol ch~ckpeulines rece~ted Srom Pak~stan to Ascochvta blight , ., in the Ascochyta hl~ghtnursery (ARN) at Hisar and In the plant growth room at IC'RISAT Asia Center, I'atancheru during 199 1-92' ..- - Disease scoreb -Chickpea line ABN Plant prowth room NARC' 9001 NARC 9002 NARC 9003 NARC 9004 NARC 9005 NARC 9006 NARC 9007 NARC 9008 NARC 9009 NARC YO10 Pb 7' 4.0 5.0 5.0 4.0 5.0 4.0 5.0 3.0 4.0 5.0 9.0 4.6 5.0 4.6 4.6 6.0 6.3 5.3 4.0 5.3 5.3 9.0 V = 0.874; R' = 0.76. bDisease score: 1 = no symptoms to 9 = killed bv the oathonen. '&scep;ible cieck. 134 Plant Disease/Vol. 79 No. 2 Fig. 2. Plant growth room showing screening of chickpea seedlings for Ascochyta blight resistance. significantly correlated (Table 2). Ascochyta blight resistance screening under field and partially controlled environment conditions has been described by several researchers (1,3,4,7), but their objectives were t o screen chickpea In the field or screenhouse. These results are more adaptive, with a fundamental need for strong correlation between the results from the plant growth room and those of the target area of crop Improvement. The results reported here and others not yet reported give reasons for confidence that the present controlled-environment plant growth room can serve a useful purpose, not only for practical screenlng. but also for studying the genetics of Ascochyta blight resistance. Limited germ plasm screening of 500 chickpea lines in the Ascochyta blight nurser) at Hisar and their reactions to disease In the plant growth room also supported the correlation between field and plant growth room screening. The facillty IS presently b e ~ n g used successfull) to screen germ plahm accessions and breeding material. LITLHATI'HE C l l E D I Ncnc. 1 I and Rcdd), M V 1'487 ('hirkpcr dlscdse, and lhclr control Pay?\ 271.270 In Ihc Chlckpcd M (' Sukcnd dnd K H S~ngh,cd* C'AB I n l t r n d l ~ o n a l W . r l l ~ n p t o r d ,l n y l a n d ? Ncnc. \' L Hawarc. M P ,and Rcdd). M b lYBl Chlckpca di\caser, reslslancc-\crccnlny trchniqucr I C ' R I S A I I n l o Hull 10 I C K I Y A I . P a l d n c h r ~ uP 0 .Andhra Pradc\h 502 124. l n d ~ r 1 Kcddy. M L S ~ n y h .K H . and Vcnc. Y I 1984 Scrcenlny t c c h n ~ q u olo1 a r i < r h y l a bllyhl u l c h l i l p c a h'agc<45-54 In A\co'h)lr l l l ~ y h .inJ t . . . H'lntcr S o n ~ n yol C h t c l p t r H (' Sukcna and K H Slngh. cd% M a r t ~ n u \N i j h o l l I)r W Junk l'uhll,hrr\. 1 hc Hopuc, Ucthcrlands 4 Utah,. H Jt.ulah~,4 ' M l t n l ~ l i l .M H and Slrdngc. K N IVW 9 q u r n l l l a t n r scrlc lor .#,\crslng t h ~ c k p c arcurlluli l u ..(\<n,< h ~ r arahlrl C'rn .I Hot hX ? 7 V - ? 7 ? h 5 Sinyh. K H . KcJd!. M \ . and Hauarc. M I' IVV! Rrccdinp IIII rchi\lnncc 10 rbruch\ln hliphl 111 rhit.lpca I'uyc\ 21-54 I n I>~scnrc K c \ ~ ~ ! a nHrcrdiny ~c i n ('hlckpcd I'roc ( q~n\ull U c r l Hrccd 1)1\ K r r ! \ l L r b u l i ('h~ckpcu K H h n p h dnd M (' 5aacna. cds 1111 ('cntri l y r i c Kc\ I ) r \ Arcdr. Alcppu, 5 ) l t u fn Irrpcro-Crsa,. A r n d Kdlscr. H' J IVV! , pl~lll l o l l u r n ~ col ~ c n i p r r a l u ~ cn,c l ~ ~ c rprrn)d. ,tyr. and ~ m ~ c u l u n i r ~ ~ n ~ c n lIUI l a l~i un nl c c l ~ oand n d c ~ r l c ~ p ~ r incl ~ ALEOL~+IJ il hliphl 1.1 c l 1 1 0 . p ~ ~ I'h+topalhailap\ X2 5Xu TVh 1 WCI\II~~. K , K,ICII~II~ICI, I ) . I pplcn, J I W r ~ g a n d ,I \axcnr. M ( ' . a n d Lahl. ( I lVVl I ) h A ( i n y r r p t \ n ~ ~ n y 4 j 1 c), /1~10~ U / , I I I wtth a ~ t ~I,CIICI ~ ~ ~ l c ~ ~ t ~ \\111hcl1c ~ ~ l t ~ ~ ~ d c ~ ~ C'UII I 9 481-4uu . . . . . Plant Diseese/February 1995 135