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Evaluation of greenhouse inoculation techniques to screen sorghum for resistance to downy mildew *

Y.D. Narayana, L.K. Mughogho & R. Bandyopadhyay Inlernational Crops Research lnstitutefor the Serrri-Arid Trol~ics (ICHISAT), Putarrc~hcru. Arttihrti Pr(~tlc.sh. 502 324. India

Rcce~vcd 20 June 1994: accepted 2 1 May 1995

Key ttords: Sor<qhuni hic,olor, Pc~roriosclc~ros~~or(i sors~tii, downy mildew. Inocul;~tlori tcchn~quc, rcslst;uicc, sorghum

Summary

Six inoculation techniques were compared for the artificial promotion of downy mildew (Pc~ronosc~lero.sl~or~~ sorghi) in sorghum. These were ( I ) sprouted seeds incubated between sporulating infected leaves, (2) sprouted sceds dipped in conidial suspension, (3) sprouted seeds sprityed with conidial suspension, (4) seedlings at plumule stitge inoculated with drops of a con.td~al suspension, ( 5 ) seedlings at plumule stage sprayed with a conidial suspension, and (6) seedling showered with conidia falling from inf'ectcd leaves. Seedlings at the one-leaf stage sprayed with a conidial suspension (6 x 105 ml- ' ) showed the highest systemic infection (100%) in the susceptible lines IS 643 a i d IS 18433. I l l is technique is effective, repeatable, and allows the deposition of a conidial suspension as :I fine mist on the entire seedling surface. In the greenhouse, the technique was used to tcst the downy mildew reaction of genotypes previously reported as resistant (< 5% incidence) in 3-4 years of field screenings. Of the 61 genotypes tested, 21 were free from downy mildew, 14 had less than 5% incidence, and the rest showed variable susceptible reactions. Therefore, the technique can be reliably and cffcctivcly used in the greenhouse to detect disease escapes and to indentify resistance.

Downy mildew, caused by Perorrosclerosporu sor%~hi (Weston & Uppal) Shaw, is a dcstruc~ive disease of sorghum (Sorghum bicolor (L.) Moench) and maize (Zru nluys L.) in cool, humid areas ofthe world (Fred- eriksen & Rcnfro, 1977; Frederiksen, 1980). The pathogen infects the roots primarily by oospores and the leaves by conidia, and reaches the meristem caus- ing systemic infection. Conidiaare produced as a white downy growthon the abaxial surface of infected leaves. Airborne conidia are the major infective propagules for secondary infection and to a limited extent for primary infection. Soilborne oospores are responsible for n~ost primary infections of seedlings in the field.

There are several options for downy mildew man- agement such as host plant resistance, chemical con-

* Approved as Journal Article No. 1667 by the International Cropr Research Institute for h e Semi-Arid Tropics (ICRISAT), Patanchcru. Andhar Pradesh. 502 324, India

trol, and cultural methods (Williams, 1984). Screening for resistance to the disease hils been carried out in the field and in the greenhouse. Anrthosur & Ilegdc ( 1979) compared several inoculation teclinrques in the lield and found the infector row method most effec- tive. Similarly, different inoculation techniques have been used in the greenhouse to tcst the resistance of sorghum genotypes to downy mildew by different workers (Craig, 1976; Jones, 1970; Schmitt & Freytag, 1974; Willianlset al., 1982). These techn~quesdiffered with respect to age of plants at inoculation, plant p,ms inoculated, method of inoculum placement, and post- inoculation incubation conditions. The relative efficacy of these techniques is not known. A greenhouse inoc- ulation technique that can induce 100% infection in susceptible controls would be useful to chcck the reli- ability of field reactions by detecting disease escapes, and in studies dealing with the genetics of resistance and pathogen variability.

The purpose of this study was to compare six inouclation techniques in the greenhouse to select a technique .that is simple, reliable, and repeatable, and that ensured infection in all inherently suscepti- ble plants. The selected technique was used to screen 'field-resistant' genotypes for downy mildew resis- tance in the greenhouse.

Materials and methods

'Ihe source of initial inoculuni was collected from downy mildew infected plants at Dharwar, Karnataka State, India. The inoculum was niultiplictl and main- tained on downy mildew susceptible sorghum cultivar DMS 652 at Palancheru where thc greenhouse cxpcri- ments were conducted. Conidia were obtained from 3- week-old systeniically infected sorghum plants main- tained in 25.4 cm-diameter pots in the greenhouse at 25 f 4O C. The plnnts were cxposcci to bright sunlight during day to allow photosynthate accuriiulation ncc- essary for abund,ult sporulation (Schmitt & Freytag, 1974). Chlorotic leaves were excised, wiped with wet absorbent cotton to remove old downy mildew coni- dia produced previously, imd wiped again using tissue paper to remove moisture from the leaf surhcc. The leaves wcre then cut into 4-5 crn lengths, and placed with their ahaxinl surface Pacing up in 9 crn-dian~eter Pctri plates litled with rnoist blotting paper on both sides. Tiie plates were incubated at 20" in thc dark for 6-7 hr for spnrulation. Conidia werc harvested by washing the sporulated leaves in chilled (So C) dis- tilled water using n camel hair brusli. The suspension was tiltered through adoublc layered of muslincloth to remove conidiophoresand other particles. The concen- tration of conidia was adjusted to 6 x 10" ml- using a hemacy tonieter. The wetting agent, 'Tween 20 (1 drop I - I ) was added to the conidial suspension before inoc- ulation.

Inoculation techn;,rucs

Sprouted seeds and emerged seedlings of the downy mildew susceptible sorghum cultivltrs IS 643 and IS 18433 (DMS 652) wcre inoculated at a time. Sprouted seeds were inoculated prior to sowing and seedlings were inoculated after emergence using various tech- niques.

Inoculation of sprouted seeds

Seeds were soaked in water for 3 hr, and placed in moist chamber for 24 hr at 28' C to sprout. Three rncthods were used to inoculate sprouted sceds: dip inoculation, spray inoculation and sandwich inocula- tion. For the dip inoculation methods, the sprouted seeds werc immersed in the conidial suspension for 5 min and the suspension was drained off. For spray inoculation method, a single layer of sprouted seeds was spread in Petri plates lined with moist filter paper, and the seeds were sprayed with a conidial suspcnsioti using iui atomiser. The sandwich inoculation method described by Safceulla (1976) was followed. In Pctri plates lined with wet tiltcr paper, sprouted sceds were placed in between two layers of infected leaf pieces in such a way that the abaxial Ie;d surtacc of both layers faced the sproutcd seeds. Sprouted seeds inoculated by these three methods werc incubated for 16 hr at 20' C in thc dark, then sown in 10 crn-diameter pots containing sterilizeti soil (Vertrsol), and niaintained in the greenhouse at 25 IL 4' C with 70-90% relative hurnidity for disease expression.

Seeds were sown in 10 cm-diameter pots containing sterilized soil (Veltisol), in a greenhouse at 25 5 4' C. At the plumule emergence stage when the lirst leaf was in the whorl. pots were transferred lo all inoc- ulation chaliiber m;~intaincd at 20 i l o C with high relativc huniidity (100%) I hour prior to inoculation. Three methods of inoculation werc used: drop inocula- tion, conidial spray. uitl conidial showering. Seedlings were drop-inoculated by placing a drop of inoculum in the whorl using a syringe (Sinph & Gopinath, 1985). Spray inoculation was done with an atomiser until the entire surface of the seedlings were covered with tine dropletsof inoculum. Forconidial showering, the outer rim of each pot with seedlings was covered with a lay- er of rnoist muslin cloth on which a layer of dctached downy mildew infected leaves was placed with abaxi- al surface facing the seedlings. On top of the infected leaves were placed 2-3 layersofwel blottingpaper. All the pots were kept in a tray containing about 1.5 ern water and covered with another tray lined with moist blotting paper. The pots were incubated overnight at 20' C to allow the infected leaves to sporulate and the conidia to drop onto the emerged seedlings. The fol- lowing morning, the muslin cloth, blotting papers, and infected leaves were removed. All the pots were trans-

Table I. Evalua~ion of doun) nirldrw ~noculation trchniqucs on tuo sorphuln pcno!)Ffl11 Ihr gn.ciihousc ;I! ICKISAT Center

Growth atapc lnoculst~on ~ n t h o d No. of rcedlinps ~nocul;~tcd l h n j nlildeu incldcncc (%)" -- IS 643 IS 18333 IS 043 IS I8433 Mcan

> Sprouted aeeds Sandwich 71 73

Dip 75 48

Spray 48 4X

Elncrped \redling\ Drop 48 48

Spray 48 47

Shouer~ng JX 41

Conuol 65 62

" Meall of three repliciitiona ot'onc tc51

ferrcd to a greenhouse and majntained at 25 5 4' C anti 70-90% relative humidiiy for the establishnient of seedlings 'and disease development. Seedlings in which water drops and water spray were used instead of the downy mildew inoculum were maintained as controls.

The six inoculation treatments and the two controls were arranged in a randomized block design with three replications, each with three pots containing about 41-75 seedlings. The number of systemically infected plantsand total plants were recorded up to 21 days after inoculation and percent downy mildew incidence was calculated. Percent data were used to perform analysis uf variance. The experiment was conducted thrice.

Evuluurion of sorghunt cultivors for r~sistance to downy t ~ ~ i l d ~ ~ t '

Sixty-one genotypes, previously reported as resistant, (< 5% downy mildew incidence) for 3-4 years in field screening at Dhruwar using infector row method (Ana- hosur & Hegde, 1979) were selected to test their reac- tions in the greenhouse. Seeds were surface steriliixd in a solution of 0. I TO mercuric chloride for 5 min, thor- oughly washed in distilled water, and planted in 10.5 cm-diameter pots containing sterilized soil (Venisol) mixed with sand and compost (2 : I : 1). Two pots, each with 50 seedlings, were maintained for each genotype. When the first leaf emerged in the whorl, the seedlings were spray-inoculated with conidial suspension (6 x lo5 ml-I), and incubated for 18 hr at 20' C and high relative humidity (90-100%) in the dark. Inoculated

seedlings were then moved to a greenhouse maintained at 25 + 4' C ~anri 7(L907crelativc huniidity for 3 weeks for disease dcvclopmcnt. The sorghum genotype DMS 652 was ni;tintaincd as susceptihlc control. The exper- iment wax contiucted in a randon1i7ed block design with two replications, each containing one pot. The greenhouse test was conducted twice. The numbers of total and diseased plants were recorded, and the percent discasc incidence was calculated. Uat;~ I'rom the two greenhouse tcsts were compared by analysis of' variance to determine the repcatability of the tech- niquc.

Results

llowny mildew incidence varied from 82 to 100% in the six inoculation methods (Table I ) . Maximum downy mildew incidence (100%) occurred when seedlings at the first leaf stage were spray-inoculated, followed by dip inoculation of spn)uted seeds (99.7%),' conidial showering (97.8%), sandwich method (95%), spray- inoculation of sprouted seeds (90%) and drop inoc- ulation (82%). Downy mildew incidences in the lat- ter two treatments were significantly less than in the seedling spray method. Roth genotypes had similar levels of downy mildew in each inoculation treatment. There was no difference in the latent period among the six inoculation methods since the symptoms appeared

Discussion 7uble 2. Number of sorghum genotypes in different claqs internal!, for pcrccnt downy rriildcw incidence in grccnhousc and lield inoculation test5

Circcnhou\c" k'icldh Total

0 0.1-3.0 3.1-5.0

" Seedling\ (lirht leal \tagc) \r~rayctl with conidial su\- ~ I I ~ I O I ~ (6 x, IIQ nil- I ),

" 1:icltl acrcclilng uvnp ~nkc to r row ~ncrhotl. Ra\ed on ~n;ixirnurndowny mlldcw ir~cidenccdat;~arllollgdle 3-4 year!, 01 lleltl hcrccnlng tcsts.

within a week after inoculation in all the methods test- ed.

Mcim downy mildew incidence in the genotypes ranged from 0-54% in 61 genotypes sprayed with conidial suspension at the sccdling stage. Twenty-one geno- types werc free from downy mildew symptoms. 12 had 0.1 to 3% systernic disease. 13 genotypes had 3.1 lo IO'lb, iuid the remaining genotypes had more than 10%1 downy mildew incidence in both greenhouse tcsts (Table 2). Although, the correlation cocflicient between the greenhouse test and the held test was sig- niticant (r = 0.68, 60 df, P < 0.01), the disease inci- dence in greenhouse tests were higher than field tests in 40 genotypes All the genotypes free from downy niildew in the greenhouse test were also disease-free in the lield; but, the reverse was not true. Downy mildew incidence in the two greenhouse tests were statistical- ly similar because the analysis of variance showed that the mean sum of squares (MS) for experiments was non significant (MS 5.70, 1 df). The correlation coefficient between the two green house tests was 0.98 (60 df, P < 0.01). This suggests that the seedling inoculation tests are repeatable.

Among the six inoculation methods evaluated in the greenhouse, conidial spray inoculation of seedlings at plumulc stage appeared most suitable for large- scale testing of sorghum genotypes for downy mildew resistance. This inoculation technique is simple repeat- able, m d effectively differentiated resistant genotypes among putatively resistant genotypes identified in the lield. Of the 61 genotypes, 21 were free from downy mildew in the greenhouse and field tests. These were IS number 133 1,2473,3546,3547,5743,7 144,7 179, 7528, 8185, 8276, 8283, 8607, 8864, 8906, 8954, 10710, 18757,22227,22228,22229, and 22230.

The three methods of seedling inoculation closely correspond to the epidemiological processes occurring in nature, wherein wind dispersed conidia deposit on young seedlings and cause infection in near-saturated environments (Williams, 1984). l i e spray inoculation method was more advantageous because it was simple, rapid, and allowed uniform deposition of conidiaon the entire seedling surface including the vicinity of grow- ing points where infection normally occurs (Jones, 1970). Schmitt & F'reytag (1974) also reported that conidial spray inoculation at seedling stage was most crficient in inducing severe downy mildew infection in corn and sorghunl. In the conidial showeri,ng method, sporulating leaves placed above the plants acted as the source of conidia, that fell on the plants by gravita- tional force. However, this mcthod was laborious, and rcquired a large number of infected leaves, carefully selected to ensure uniform sporulation during incuba- tion. Inspite of utmost precaution, some seedlings may not receive inoculum if the leaves imniediately above them do not sporulate well. Craig (1976) avoided the latter disadvantage by devising an elaborate inocula- tion chamber to permit uniform air flow for consistent conidial deposition. Drop inoculation was also labo- rious and fraught with chances of inoculum loss by dripping of inoculum droplets from the erect surfaces of young seedlings that lacked unfolded leaves at the time of inoculation. The methods used to inoculate sprouted seeds do not represent what happens under natural conditions because sprouted seeds are not nor- mally exposed to conidial inoculum.

Conidial spray inoculation of young seedlings of 6 I putatively resistant genotypes resulted in varying levels of susceptibility (0-5490) in the greenhouse, whereas the same genotypes under tield screening using the infector row method had shown < 5% susceptibility over a period of 3-4 years. Screening for resistance

to downy mildew in the field is essential to evaluate large numbers of genotypes, and to evaluate agro- nomic traits. It is often carried out using an infector row technique wherein conidia produced on the infect- ed plants are dispersed by wind to infect test plants at susceptible stage. Production of inoculum is often determined by temperature and humidity which may not be always favorable for sporulation and infection. leading to disease escapes. The seedling spray inocu- lation method in the greenhouse achieved 100% dis- ease in susceptible control and thus assured accura- cy in detecting disease escapes in putatively resistant genotypes identified in the field. Alternatively, geno- types can be initially screened in the greenhouse, and selected plants can then be tested in the field for the adaptability of the resistance trait (Keddy et al., 1902). This technique would be more effective and economic in screening large numbers of breeding material in 3 resistance brceding programs.

References

Craig. J.. 1976. An ~nwulat ion tcchnique for identifying resistance to downy mildew. Plant Dis. Rept. 60: 350-352.

Frederilisen. K.A.. 1980. Sorghum downy mildew in h e United States: Ovcrvicu~ and outl(n)k. Plant Dis. 64: 903-08.

FrrdcriLsen. R.A. R: B.L. Kcnfro. 1977. (ilohal stlitus of maize downy mildew. Ann. Rev. Pfiylopath. IS: 249-275,

Jones. B.1 ... 1970. A simple techtiique ol'incxuli~tlng sorghuni w i h S(~/erosporu ~rjr#/ti using conidii~ 3s intxuluni. I'liin~ Dis. Kcpt. 54: 603-604.

Rcddy. B.V.S., L.K. hluglrogho. Y.1). Nnreyilna. K.D. NiccxJeniu\ & J.W. Stenhouse. 1992. luheritnncc pdttcm of tlowny ~il i ldcw rcsi\tance In adviuicctl generation of sorghuln. Ann. Appl. Biol. 121: 249-255.

Sal'eeulla, K.M.. 1976. Biology ;~nrl control of downy niildews of pearl nli l lc~, sorghum and li~igcr nllllct. Wedey Rcss. 304 pp.

Sch~n~tt. C.G. X: K.E. Frcqtiig. 1974. Quantitative tcchnique for inoculatitigcom and sorghum will) conidia ol'.Sr~lcro.\porusor~hr. Plant Dis. Rept. SX: 825-829.

Singh. S.D. R: R. Gopinath. 1985. A scedhng ~noculation technique lor tletecting downy ~l i i ldcw rcsistancc in par1 millet. Plant I)i\. 68: 582-584,

Wil l~at~lr . R.J.. 1984. Ilowny mildew ol' tropical cereals. Adv, in Plant I'ath. 2: 1 -10.1.

Willinnis. H.J.. S.R.S. Dangc. L.K. MuphoghoR: K.N. Hao. 19x2. lderitilication of QL.3 \orphuni, a source of rcsistancu to Per. ono.~clcro.cpomc;or,qhi. Plittil Di\. 06: 807--809.

Allaho$ur. K.H. Xc R.K. Hcgde. 1979. Assessment o f the tech~~lque\ u\ed in \creenirig grnotyp'sto downy mildew. Mysore J. Agric. Sci. 13: 449-45 1 .

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