Temperature-Induced Suppression of Alternaria Leaf Spot of Cotton in Arizona

P. J. COTTY, Department of Plant Pathology. University of Arizona. Yuma Agricultural Center, Yuma 85364

ABSTRACTCouy. r. J. 19117, ,'cmpc... tur-e-lnductd suppfcs.sioll of Al'(turia leilfspot ofcotton in Ari~on•.Plant Di$tase 71:IIJI·II40.

lainn (ormation by AlurnQritl rnauosportl. the a1u$lI1 agenl of Allernan. leaf 6pot of colton(GoIIJ'pium /)Q,bluhnvJ. is innuenced by fhe tcmpcnlurc at ..-hich pia nu are held .ftcr inf«lioD.Fen)' 10 100% rwucliolU in lesion number otnlrrw on c:otlon planls inccubaled a, CIC\'altGlcmperalurcsc:omparcd ..·;th plants m;ainl;a;Md :lr30C.lC'Sion formalion was reduced more.lbtl7(1% .rtel upon",: 10 4J.j C (01 2 hr. RcsullS indicate IMI daily tcmpcr.llture mu.im.a may beimponanl in limiti". Alterun.a leaf SPOI in Arizonl and may plruallytxplain increased disease5e\'Crir)' in cenl 1"11 Arizona daring counn seloSOr15 in whkh daily temptr.u I,Irt ma:o.ima ..~ ~lIti\,tly

low. The innuc","o(h'lh rernpe:ralurtOn lhe p:nho8tn in \'ilrowaJ 1olsoRuditd. Germ tubes orA.mtJuOlportJ Upl05i~ly l)'S("d on potalo-duuose I,ar (I'OA) IMuNled al 42-46 C. "fler 6 hral42.43 5. Irwl4ll C. rt5p«li\·dy. 7. S2. and 82% of tilt gtrm tubes lysed. Sport \'iabililY on PO"declined 6S% aftcr 4 hr al 46 C but r<:mainc:d sltble for 24 hr II 42 C. Results provide indir«tevidcnee thatlcrm lUbe lysis and reduced spore Vil~lil)' may be m«hanl!ims of tempcraturt­mediated reduclion in Ic!iion number. lIoll'cver. lither temperature.induced .lterations in Ihehost-pathogen inleraction may be equally important

Table I. frtquency of vllriousdilily lcmptfllturt ma.Jlima al Ca$a Grande Nationill Monument.C.na Grande. A'Z:

'0. da)'$ Itmptrallitt u:ertd~

Montb YR' J7C <GC 43C "C

MIlY 1984 22 " 1 01985 "

, 0 0J"~ '98' 26 " 2 0

19S5 26 " ",

July '''' 2J IJ • 0

'98' 29 28 16 1AUIUSt '98' " IJ 2 0

19l1S JO 28 , 2September ".. " J 0 0

19S5 ", 0 0

May to Seplember 19114 '" " " 019S5 111 '" .. "•Dau from National Oceanie .lId Almo$pheric: Adminislr.uion. U.S. Dl:psflnlCnl of Commerce.

confirmed with the previously described(4.5) isolates C·22. CB-I. C·) (ATCC58174). and C·IO (ATCC 58175).Sporulating cultures of fungal isolatcswere maintained on a modiftcd V-8medium (5) containing 5% V-8 vegelablejuitt (v/v) and 2% agar (w/v) at 27 Cunder 5.()()().lux nuorcsccntliahl on a 12­hr diurnal light cycle. For long-termSIOrage. 3-mm-diametcr :tgar plugs ofsporulating cultures were. maintained atroom temperature in 25-ml vialseonlain­ing 5 ml of sterile distilled ...... ter.

[HeelS or IfmperatUft on lesionrormalion. To test the crfl"Cts of hightemperatures after initial infection onsubsequent lesion development, plantswere exposed to variuus tempcratures for6 hr shortly aftcr infection with A.macr()!fporu. Colton plants«(i. burbudensecv. Pima S-6) were grown in II greenhousein '50-ml pots conllining a modifiedV.C. mix (pt:at moss. sand. \'crmiculite(8:6:IJ plus 21 g KNO,. 21 g K)SO., 69 gtre.ble superphosphate. )1 I g dolomitelime, 104 g CaCO" and 10 g CuSO. percubic meter) and were fertilized with 50ml, 2.000 ppm, Miracle-Gro every 8-10days. Plants at the. four- to siX-leaf stage(30-35 days old) were Spr1lyed to runoffwith suspensions containing 3,000-4.000spores per milliliter in 0.001% Triton X·100. Ihen immediatdy placed in anunilluminated humidity cha mber (rela ti \'ehumidity 100%) maintained at 26-28 Cfor 15 hr. A duration of 15 hr was usedbecause it resulted in moderate diseaselevels in the controls. Dew periodstypically have shorter durations in thedescrt valleys of Arizona. Subscquenlly.plants were removed from the humiditychamber. allowed 10 dry (about l.5 hr),and then incubated al either 30, 39. 42. or45 C for 6 hr before being placed on thegreenhousc bench. After 5days. alllel\'es

Outbreaks of ALS during 1982-1984indicate yirulent isolates arc present inArizona (4.8). and recent acreageincreases or susceptible eullivars maycontribute to increascd ALS levels (4).However, subsidence of diseasc preuurein 1985 as succptible acreage increasedmay indicale that environment plays animponam role. Allhough temperatules(25-30 C) fa\'oring infection of colton byA. marrospora (2) occur daily during thccotton season in Arizona, daily temper·:uurc: maxima u:cceding 40 Carecommon (Table I).

The current study was undertaken 10determine whethershort-term exposuresto high temperatures afler infection, asoccur in the: desert V1Illeys of Arizona.innuencedisease incidence. The effects ofe1evlted temperature on the fungus invilro also were studicd.

MATERIALS AND METHODSCullurU. A. maCfospora isolate C-20

from G. barbaJensecoliected in Safford.AZ, in 1985 was used in all in vivostUdies. In vitro phenomena were

Alternaria leaf spot (ALS) of cottoncaused by A Irernllrill ma('foJpora Zimm.occurs in most cotton-growing areas ofthe world (3.7). Although most canonspecies are susceptible to the pal hogen,the predominant species grown in theUnited States, (joSJ)pium hirJulum L. ishighly resistant (7). However, extra-long­staple Pima colton (G. barbadrnsr L.) ishighl)' susceplible. In Israc:l, ALSreduced yields of the G. barbadrnsrcuhivar rima S-5 up to 25% (3). PimaCOllon has been grown commercially inArizona ror many years with only minorlevels of AI.S in mosl years (IO).However, in 1982. 1983. and 1984.increased rainfall and reduced dailytemperature maxima occurred andmodrrale to hea\'y levels of ALSdeveloped in cenlral and soulheasternArizona eOllon (4.8). The role of frccmoisture in infection by A. macrosporahas been well documented (2). and rainpatterns may be involVed in the increaseddisease pressure during 1982-1984.However, dews arc sufficient to supportsevere epidemics of AlS during dryIsraeli summers. and Bashi ct al (2)questioned Why ALS is not a perennialproblem in the United States. Theseauthors (2) proposed three explanations:1) limited acrcage cropped to sensitivevarielies, 2) absence of virulent slrains ofA. ma(fospora, or 3) an unfavorableenvironment for disease development.

AtttPltd for p"bl>eat,Oft 2. Alllusr J981 hubrnillcdIOf e,",ro..'" pr~;"ll.

P,coc... add,~ 0( I"thor; U.S. Departme..t ofA.neah",e, A,ricult"'ll Rnnrch Sn~icc.

Southc". R~poIIIl Rcocatch CmICf. po. Do"19681. New Oflclns.LA 70179.

Journal Seun P ' .1}99 of tbe "'''lonAlrKuJt"nol E:lpcri Statio... T.......

o 1981 The Amerlean PhytOPilthotogJC41 $oeiety

1138 Plant Dls8aS8lVol. 11 No. 12

Table 1. Number of lesions on Alternaria maerospora-illfectcd collon pIa nlS mai Dtained Qt varioustemperature. for 6 hr immediately after infection

xpulmrntno.

I2

456

o 1_0.030.49 ± 0.33

0.0 0.06O. _0.19

4S C

0.00.02 ± 0.02

Rrducl1on~

(%)

100.09.292.17 .171.745.0

Table 3. Number of lesions on AlternariamaCfospora·infected cotton planlS maintainedat 43.5 C for 0.2,4. or 6 hr immediately afterinfeaion

6

•Experimtnts contained tWO lreaunents with (our replicate. Differences in the number of Ie ions'thin e perimen an: igniflCaDl (P= 0.01) by 8lUll i of variance ucept in e perimenl6.

Perunt reduction in the numbc:rof~ionsformed t t· temperaturescomparcd ith i ubationat 3D C.

'Treatments contained three replicates.'umbers in the me olumn follo\\ed b theme letter are not si oirt ntJy dirrerenl (P

= 0.01) by uke' tudentil.ed range test.

formed (Table ).Elevated temperatures also influenced

fungal behavior in vitro. Germ tubes offive A. macrospora i olates expo ed to41-46 C Iy ed readily on PDA. Lysisappeared explosive with cellular contentsextruded through lateral breaks in thegerm tubes. Lysed germ tubes appearedpartially or completely vacant. whereasintact germ tubes contained relativelyevenly distributed cytopla m tbalretained cotlon blue. Germ tube lysisincreased with temperature (Fig. I) 0

thai 7, 52. and 82% of the germ tubesunderwent Iy j within hr at 42. 43.5.and 46 C. respectivel .

pore viability de r don PDA withincreases in temperalure from 42 to 46 Cduring the 72-hr test period (Fig. 2). Atthe highest temperature. C. pore

'abilit decrea ed 65 within 4 hr,whereas at42 , pore viability remainedstable for 24 hr.

100

90

80 ,J-----j-.:::> 70~~ ,.... ,QJ 60 J/..c::J....E 50~

8-.... ,40 IC ,

~u ,... ,Cll

30 ,0- ,

20 /Y ",f",.,.

10~,/ ,_.-t'.

0 -------....-. _.-J--

pore . 'xperiment ere performedtwice and replicated three times.

RE TSExpo ure of colton plants infected

with A. macrospora to 39, 42, 43.5, or 45for 6 hr resulted in reductions in the

number of lesion formed compared withplants maintained at 30 for that period(Tables 2 and 3). The highest temperaturetested, 45 C, resulted in 99-100%reductions in lesion number per unit arearelative to the contTol . Lower temper­ature were less effective in limiting lesionde elopmen Howe er e en incubationat 39 C for 6 br resulted in 40-7 fewerIe. ion . Lesion formation as reducede en ben plant ere expo ed toele ted temperatures for period shanerthan 6 hr (Table 3). Expo ure for 2 and 4hr resulted in significant P < 0.01)reduction in the number of lesion

456Time (hrl

f'il' 1, Percentage of Alternaria macrospora germ lubes lysed at 41 (- . -J. 43.S C (- - -). and46 C (-) after incubation on potato-delliro e agar for 0-72 hr.

were l:xci ed, pbotocopied and thelesions on each leafcounted. Leaf uTfaceareas were calculated by video imageanalysis. ach experiment contained t 0

temperature treatments including thecontrol treatment, 30 C, which is in theoptimal range for host colonization bythis fungus (2). Experiments wereperformed twice and replicated fourtimes. Replicates consisted of individualplants.

In another experiment, infected plantswere maintained at 43.5 C for 2 4 or 6 hrand compared with control plantSmaintained at 30 C for 6 hr. Plan in the2- and 4-hr trcatm nts were transferredto the 30 C chamber at the appropriatetime for the remainder of the 6--hr period.This e:w.:periment w repeated once withthree replicates per treatment.

In itro tudit. To assess sporesurvival and germ tube beha ior in itro,por from 5-day-<lld culture of A.

macrospora ere upended in distilledwater to a fin I concentration of about3 OOO/ml. Eight 10-$11 aliquolS of pore

uspension were applied to the potato­dextro eagar (PDA) urface in each 9­em-diameter pia tic petri dish andincubated at 27 . After I hr, all porehad produced three to 10 germ tubes.Plates were then incubated at the testtemperature (38,41,43.5, or 46.0 C) for 2,4, 6, 8, 10, or 12 hr. At the end of eachperiod, spores and germ tubes were fixedand tained on the agar surface with 0.1%colton blue in laclophenol. Pieces of agarcontaining spores wer'C then removedfrom four location on each ofl 0 platesfrom eacb treatment and examinedmkro copieally 600X). Germ tubesexplosively lysed at temperatures higherthan 3 ererorc, the percentage ofgerm tube Iy . determined for eachtreatmenL E periment ere replicatedeight times and performed twice.

Spore survival at ele ted temperatureson PD detcrmined b spreadingSO-SO spore.! over the agar surface ofeach PDA plate and incubating at 42­43.5, or 46 for 4, 8, 12,24. or 48 hr.After exposure to elevated temperatures.plate were incubated at 27 C for 24, 4S,and 96 hr and e ami ned with a dissectingmicro. cope (40X) to determine thepercentage of germinating and growing

Plant Disease/December 1987 1139

100~ "I "-I

'1"-

80\I

\'"I

> Ic: 60 I \::J I'" \'"~ I

\0\0-

'" I- 40 Ic:\'" Iu

~ \'""- \ \\

\20 \ \\

I\ \\\

0 b "0 8 24 48 72

Time (h rl,,'il· 2. PCfC(nUlee of Aff~rnQritI"'QUlJsPO'Q$PO~ iurvivinl incub.a1ion on pollto-dulrOliC: apfat 41 C(-' 1,43.5 C(-- ~l, and 46 C(-I for 0-72 hr.

,,.,, Plant OiseaselVol. 71 No. 12

DISCUSSIONFOrly-eight hours lapse between

infection ofcotton by A. marwspDrQ andexpf"tssion of Ihe initial ALS symptoms(2,4). This stage in disease de'·e1opmenlhas not been studied in detail. but it isapparently a period of host tissuecolonization (2). Results of the currentnudy indicate that this early pre­symptomatic stage in disease developmentis highly sus«:ptible to disruption byelevated temperatures (39-46 C). Whenplants with presymptomatic infectionsarc incubated at43.5(110 F) for just 2 hr.there: is at leaSI a 70% reduction in thenumber of lesions formed (Table 3).Furthermore, the lcvel of diseasesuppression increases with tcmperature(Table 2) and exposure time (Table 3),Daily temperature maxima between 39and 46 C are typical in central Ari7.onaduring the cotton season (Table I). Thus,although the presence of free moisture(dew) and moderate temperatur« atnight may favor infection in the Arizonadesert (2), elevated temperatures duringthe day following infection may limitdevelopment of Al..S symptoms. Theinfluence of daily temperature maximamay be particularly important in Arizonaearly in the season (June and July) before

canopy closure. when hoI. dry winds andwater stress arc common. Establishmentof the disease ea.rly in the season isconsidered an important componentleading 10 severe ALS epidemics in Israc:l(2).

In ttntral Arizona. temperatures in theoptimal range for infeclion by A.mQcrosporQ (20-30 C) occur most nighuin years with either high or low ALSincidences. Ho.....ever. daily temperaturemaxima .....ere lower during 1984, a year inwhich ALS was heavy, than in 1995,when disease pressure greatly declined(Table I). DuringJunc and July 1985,thedaily temperature maximum exceeded 43C351imes, whcrcasin 1994, temperaturesonly exceeded 43 C SilC timu. Diseasedevelopment is rapidly disrupted at 43.5C (Table 3). Therefore, low dailytempe:nllun: maxima may have contrib­uted to the prevalence of AL$ in 1984and may be indicative of periods favoringdiselilse development in Ari:wna_ Thissupporu the suggestion (2) that environ·mental conSlraintslimit ALS in Arizonain most years.

Rapid lysis of A. mQrrosporQ germtubes in vitro atlemperatuf"ts higher than38 C (Fig. I) may partly explain thereduction in lesion formation caused by

exposing infected plants 10 thesetemperatures. Germ tube I)'sis increascswith increased temperature and uposuretime (Fig. I). Thus, germ tube lysis maybe a mechanism through whichtemperature-mediated reductions inlesion establishment result. However, theobsernd phenomenon may be due toother alterations in the hostfpathogeninteraction.

Spore viability also declined on PDAat temperatures higher than ]g C.Ho.....ever, viability reduction was tooslow to be significanl in situ except attemperatures higher than 45 C. A Irtrnariaspores are less sensitive to temperature ina desiccated state, which probablyreflects the conditions under which thesepathogens are typically dispersed indeserts (9). Therefore, high dailytemperalure maxima probably do notsignificantly reduce the inoculumpotential of A. marrosporQ in theArizona desert valleys.

Temperatun:s tested in the currentstudy reflect typical daily temperatun:maxima oa:ufTing in central Arizonaduring June and July (Table I).However, actuallc:afsurface tem pcraturesin the field will vary greatly dependingupon variables such as light, hostnitrogen metabolism. water stress,position within the canopy, and relativehumidity; these faclors also independentlyinfluence the incidence and severity ofdisease (I). Light-dependent host defenscsinfluence the outcome of A IltrnQ,iQfhostinteractions (6). Beeause high lightinlensitin oceu r simultaneously wit bdaily temperature maxima, these factorsmay aCI in conttrt against the pathogen.

ACKNOWLEDGMENTI 'Irish 10 lhan\ J. P. EVlIfti fOf auisUonu ";11l11lo:

wicko ima~ anall'lil.

I.ITf.RAlURE CITEDI. AlTlOi. G. N. 1975. Pllnl Palh<llnsy. AQdemil:

I'IUS, Sew York. 71)) pp.2. Ilalhi. Ii.. ROlem. J., Pinn><:hmidl. H., and

Kranz, J. 19U. tnOuente of tonlrolJeduvironmenl nd a,e on developmenl ofAI,r""". "''"''''f''O''' and On lheddio. ofleaves 01 eotl0ll. f'll)'lopl,holo&)' 73:114S-1147.

3 Bo.Plt E., Sal:hI., Y.• and Rotrfll, J. 19111.Rdalion...ips IoeIIlU1l dika..e and yield in«lIlon flddl affecud by AlltrM'~ m«roJPCU.Ph)10plfasllica II :19-91,

4. Cony, P. J. 19n. COIIOIl (lilt"",. Ill1Oep1ihilily10 AlIe'lIa";" ItafipoL P'lam 0._. 71:OllO-OOO.

S. COlly. P J .. and M<saahi, I. J. 19U. ZinRiolp(OdllnJOll by~~ Phytopalho!o&Y74:7U-7ll1.

~, Couy. P. J., and M<sa"'i. I. J. 1'llS. Effttl ofI;lh, on ,he beh.lviOf of AI'r"'",~ 'IIIftffl. invilrO and in vivo. Phylopa'''''loty 7S:366-370.

7. Jonn. G. II. 1925. An All"",,,;. di,eo<w: or 1MCOHon pllnl. Ann. lUll. 42:93S·947.

I. OIJen. M. W.• and COlly, 1'. J. 1986. Conlrol orAl1ernarillear SPOI of a"Jsypiu", />D""HI,n".Paeel 21.28 in; P'O(. Ikllwlde COllon I'rod.Rei. Conf.. Lai Vep.l. NV. S24 pp.

9. Rotem.J.I96ll. Thefmoufoph)'lkpropenicoofAI,rrrtflrV porri f. sp. """'ni. Ph)'topa,holon».1284-1287.

10. S,,_... 1\. 8..S•. 1969. Disetielohllceuluntedplanll of 'M Soulhwnr_ UlliWnil)' of AriwnaPfQ.l., T~ll.)90 pp.