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Effects of conidial concentration and stigma wetness period on infection by the sorghum ergot pathogen Claviceps africana

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The effect of conidial concentration and length of stigma wetness period on infection of sorghum spikelets by Claviceps africana was studied in a controlled environment experiment. Aspore concentration of at least 106 conidia/mL was required for
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  Effects of conidial concentration and stigma wetness periodon infection by the sorghum ergot pathogen  Claviceps africana  S. A. Bhuiyan A , E , M. J. Ryley  C , V. J. Galea B , D. Tay  D and A. T. Lisle  B A BSES Limited, Private Bag 4, Bundaberg DC, Qld 4670, Australia. B School of Agronomy and Horticulture, The University of Queensland, Gatton, Qld 4345, Australia. C Queensland Department of Primary Industries and Fisheries, PO Box 102, Toowoomba, Qld 4350, Australia. D Ornamental Plant Germplasm Centre, The Ohio State University, Columbus, OH 43210, USA. E Corresponding author. Email: sbhuiyan@bses.org.au Abstract.  The effect of conidial concentration and length of stigma wetness period on infection of sorghum spikelets by Clavicepsafricana wasstudiedinacontrolledenvironmentexperiment.Asporeconcentrationofatleast10 6 conidia/mLwasrequiredformaximuminfectiononsorghumpanicles.Maximuminfectionoccurredduringastigmawetnessperiodbetween4.5 and 6 h. Approximately 11  –  15% infection occurred when stigmas had been dried immediately after inoculation.This study demonstrated that the stigma wetness is an important parameter in sorghum ergot epidemiology.Sorghum ergot, caused by  Claviceps africana  Frederickson,Mantle & de Milliano, is an important disease of sorghum[ Sorghum bicolor   (L.) Moench] (Bandyopadhyay  et al  . 1998). C. africana  was con 󿬁 ned to Africa until the mid 1990s, but since1995ithasbeenreportedinNorthandSouthAmerica(Reis etal  .1996;Isakeit  etal  .1998),Australia(Ryley etal  .1996),andIndia(BogoandMantle1999).Malesterile(A-)linesinF 1 hybridseed  production are especially at risk  (Bandyopadhyay  et al  . 1998). C. africana  colonises the ovaries of its hosts after conidiagerminate on the stigmas, styles or ovary wall, and hyphaegrow into the ovary (Bandyopadhyay  et al  . 1998). The ovaryisreplacedbyasphacelium,whichmaydevelopintoasclerotiumcontainingalkaloidsthataretoxictoanimalsifconsumed(Blaney et al  . 2000). Macroconidia are produced at the apices of short conidiophores on the surfaces of the sphacelia (Bandyopadhyay etal  .1998).Approximately7  –  8daysafter invasionoftheovary,clear, sticky drops of honeydew containing macroconidia and microconidiaoozefrominfectedspikelets(Bandyopadhyay etal  .1998). Under conditions of high relative humidity (RH), manymacroconidia of   C. africana  near the surface of the honeydewgerminate to produce sterigmata above its surface and under suitableconditionsasecondaryconidiumdevelopsattheapexof each sterigma (Bandyopadhyay  et al  . 1998). It is believed that these airborne secondary conidia are responsible for the rapid spread of   C. africana  (Frederickson  et al  .1993; Bandyopadhyay et al  . 1998).The window of infection by conidia of   C. africana  is from 󿬂 ower opening at the onset of anthesis until fertilisation (Futrelland Webster 1965; Bandyopadhyay  et al  . 1998). Pollinated ovaries are rarely infected (Futrell and Webster 1965;Musabyimana  et al  . 1995). Environmental factors such astemperature, RH and rainfall have a signi 󿬁 cant impact onergot severity, not only by reducing the viability, release and germinationofpollen(Quinby1958;McLarenandWehner1992;Tonapi  et al  . 2002; Ryley 2005), but also through their effectson the production, transport and infection of secondary conidia(Frederickson  et al  . 1989; Bandyopadhyay  et al  . 1998; Tonapi et al  . 2002). Temperatures near 20  C are considered to beoptimum for maximum ergot infection (Anahosur and Patil1982; McLaren and Wehner 1990; Ryley  et al  . 2002), withseverity decreasing as the temperature at inoculation increasesabove20  C(WorknehandRush2006).Rainfallduring 󿬂 oweringhas also been associated with high ergot infection levels(Ryley  et al  . 2002; Workneh  et al  . 2006), but there is someevidence that rainfall is not necessary for successful ergot infection if RH during  󿬂 owering is high. Futrell and Webster (1966)consideredthataRHof  > 90%wasnecessaryforoptimumergotdevelopment,whileWorknehandRush(2006)reportedthat in inoculated   󿬁 eld trials, infection increased in a linear or exponential fashion, depending on the inoculum concentration,asRHatthetimeofinoculationincreased.Theyalsofoundthatalow level of infection occurred when RH at inoculation was20  –  30%, suggesting that the minimum RH needed for infectionis much lower than previously thought. Molefe (1975) reported that panicles inoculated with  Sphacelia sorghi  and incubated for 20 h at high humidity and 20  –  25  C developed honeydewwithin 7 days, and also observed that in the year of high rainfall(  750 mm) there was a high incidence of ergot (25  –  90%).Thakur   et al  . (1991) reported that 8 h of stigma wetness wasrequired to obtain 70  –  72% of infected spikelets by  Claviceps fusiformis  in pearl millet at temperatures below 20  C.Studies which have involved inoculation of   Sorghum  species byconidia C.africana haveusedaqueoussuspensionsofconidia,atconcentrationsrangingfromunquanti 󿬁 edupto10 6 conidia/mL(Musabyimana  et al  . 1995; McLaren and Flett 1998; Worknehand Rush 2006). The results of Workneh and Rush (2006) CSIRO PUBLISHING  Australasian Plant Pathology , 2009,  38 , 496  –  499 www.publish.csiro.au/journals/app   Australasian Plant Pathology Society 2009 10.1071/AP09032 0815-3191/09/050496  suggest that ergot infection increased as the conidialconcentration increased, and that the concentration in 󿬂 uenced therelationshipbetweentemperatureandRH,andergotseverity.There is little reported information on the effect of free water onthe stigma on infection of sorghum by ergot pathogens. Most of the relevant literature is based on anecdotal evidence and observations.Theobjectivesoftheresearchreportedinthispaper were to determine, under controlled conditions: (i) the effects of different conidial concentrations of   C. africana ; and (ii) theeffects of stigma wetness period on infection.Foreachexperiment, 󿬁 veseedsofamalesterile S.bicolor  line(Paci 󿬁 c Seeds, Toowoomba, Qld, Australia, proprietary line)were sown in a potting mixture of pine bark, saw dust and sand (2:2:1) in 20-cm-diameter pots in a glasshouse. Theseedlings were thinned to two per pot when the plants were~20 cm high. The plants were watered twice daily and fertilised with Aquasol (Hortico, Revesby, NSW, Australia) once a week.When~50%ofthespikeletsoneachpaniclehad  󿬂 owered,the potted plants were transferred to a growth cabinet maintained at 20  1  C with a 12-h dark and light period. At ~50% anthesis,upper branches with previously  󿬂 owered spikelets, and lower  branches with spikelets which had not yet   󿬂 owered, wereremoved from the panicles. Fresh honeydew, containing bothmacroconidiaandsecondaryconidia,wascollectedfrominfected  panicles on plants of several commercial grain sorghum hybridsgrowing in a  󿬁 eld at the Gatton Research Station, Gatton, Qld (~27  55 0 S, 152  33 0 E). A range of conidial concentrations(10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 conidia/mL) were prepared by mixing the honeydew with sterile deionised water,adjusting the original solution to 10 7 conidia/mL using ahaemocytometer, then preparing a serial dilution series. Four randomlyselected  󿬂 oweringpaniclesweresprayedusingasterilehand sprayer until runoff, with each of the conidial suspensions.Anotherfourpaniclesweresprayedwithsteriledeionisedwatertoserve as controls. After inoculation each panicle was covered immediately with a brown paper bag for 48 h. Both RH and temperatureintheglasshouseweremonitoredandrecordedwithadata logger (Datataker, Model DT50, Rowville, Vic., Australia).Fifteendaysaftertheinoculationthetotalnumbersofspikeletsandthenumbersofspikeletscontainingsphaceliaoneachpaniclewere counted, the percentage infected spikelets in each paniclewere determined, and the mean percentage infected spikelets for each treatment calculated.Thein 󿬂 uenceofdifferentdurationsoffreewateronstigmaoninfection(0,2,4,8,12,24,48and72h)wasstudied.Asdescribed  previously,16potseachcontainingtwoplantsweretransferredtoa growth cabinet when the panicles had reached 50% anthesis.All panicles were sprayed to runoff with a standard conidialsuspension (10 6 conidia/mL) using a sterile hand sprayer.Immediately after spraying all plants were covered with alarge plastic bag (240 L, 147    113 cm, Multix, Multix PtyLtd,Clayton,Vic.,Australia),exceptforfourplantsonwhichthestigmas were dried immediately after inoculation by directingheated air (low heat setting) from a domestic hair dryer (RonsonHeatweave1000,RonsonPtyLtd,Sydney,NSW,Australia)ontothepaniclesfor10  –  15min.Theseplantswerethenplacedbackinthegrowthcabinet.After2,4,8,12,24,48and72hfourrandomlyselected panicles were removed from the plastic bag and dried asdescribed above. The temperature of the growth cabinet wasmaintained at 20  1  C and the RH outside and inside the plastic bag were recorded using humidity sensors [TinyTag Plus,Gemini Data Loggers Ltd (Chichester, UK)]. Plants wereincubated in the growth cabinet for 9 days, then transferred toa glasshouse. Fifteen days after inoculation the mean percentageof infected spikelets for each inoculated treatment wasdetermined as described earlier.A second experiment was conducted using stigma wetness periods of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,and 6.0 h duration. The procedures used for this experiment were the same as those of the  󿬁 rst experiment.The data were analysed initially using the SAS version 6.12(SAS Institute, Cary, NC, USA) general linear model procedure.Response functions were then  󿬁 tted to the mean data by non-linear regressions.Alogisticmodelwasusedtodescribetheeffectofthedurationof high humidity on ergot infection (  I  ) as:  I   ¼  M  1 þ e  k  ð h   HM  Þ  ð 1 Þ where  I   = % infected spikelets/panicle,  M   = estimated maximuminfection, k  =estimatedrateofinfection,  HM  =estimatedmedianinfectiontime,i.e.wheninfection=  M  /  2 ,and  h =durationofhighhumidity (h).There was a clear relationship between the concentrationof conidia and % infected spikelets, with the highest levelsof infection ( > 75% infected spikelets/panicles) occurring at concentrations   10 6 conidia/mL (Fig. 1). The mean value of % infected spikelets/panicle increased exponentially up to10 6 conidia/mL as the log 10  conidial concentration increased;at 10 5 conidia/mL the mean value of % infected spikelets was~50% (Fig. 1). A suspension of 10 6 conidia/mL was used in theexperiments on the relationship between the duration of stigmawetness period and ergot infection levels.During the  󿬁 rst experiment the RH inside the growth cabinet was 81  2% for the  󿬁 rst 5 h, 100% for the following hour, then 󿬂 uctuated between 42 and 80% for the remainder of theexperiment. RH inside the bag was 80    2% for the  󿬁 rst 6 h, Log 10  concentration (conidia/mL) 0 2 4 6 8    %    i  n   f  e  c   t  e   d  s  p   i   k  e   l  e   t  s   /  p  a  n   i  c   l  e 020406080100 Fig. 1.  Effect of concentration of   Claviceps africana  conidia on infectionof sorghum spikelets at 20  1  C in a growth cabinet. Error bars denote thestandard error of the means. Conidial concentration, relative humidity and ergot infection  Australasian Plant Pathology  497  then 98    2% for the remainder of the experiment. In the second experiment, the RH inside the growth cabinet was 80  –  100% for the 󿬁 rst10h, 󿬂 uctuatingbetween48and80%forthenext8days,then increasing to 100% for the remainder of the experiment.The RH inside the plastic bag was 98    1% for the entire period.The interaction between the stigma wetness period and  percentage infected spikelets/panicle was highly signi 󿬁 cant (  P   <  0.001) in both experiments. In the  󿬁 rst experiment, ~57%of the spikelets were infected after 2 h of incubation under thehigh RH conditions (Fig. 2). After 12 h incubation 78% of spikelets/panicles were infected, with infection levels at longer incubation periods ranging from 76 to 86%. The  󿬁 tted curvesuggests that maximum infection occurred after 6 h and that theinfectionleveldidnotchangewithincreasingdurationsofstigmawetness period. When stigmas were dried immediately after inoculation (0 h on Fig. 2), 15% of the inoculated spikeletswere infected.The effect of the stigma wetness period on % infected spikelets/panicle can be described by the following equation,  I   ¼  79 : 4 = ½ 1  þ  e  1 : 04 ð h  1 : 31 Þ  ð  R 2 ¼  0 : 96 Þ ð 2 Þ where  I   = % infected spikelets/panicle and   h  = duration of highhumidity (h).In the second experiment, 11% of spikelets were infected when stigmas had been dried immediately after inoculation. Thevalueof%infectedspikelets/panicle(  P  < 0.001)increasedasthestigma wetness period increased, reaching its highest value at 4.5 h (Fig. 3). The following equation represents the 󿬁 tted curve,  I   ¼  62 : 3 = ½ 1  þ  e  0 : 63 ð h  2 : 7 Þ  ð  R 2 ¼  0 : 95 Þ ð 3 Þ where  I   = % infected spikelets/panicle and   h  = duration of highhumidity (h).Our   󿬁 ndings indicate that a concentration of 10 6 conidia/mLwas optimum for infection of the male sterile line used inthis study, and that higher concentrations did not result insigni 󿬁 cantly higher infection levels. Conidial concentrations of ~10 6 conidia/mL were used by Tegegne  et al  . (1994),Musabyimana  et al  . (1995) and Reed   et al  . (2002) in their studies to identify resistant germplasm of   Sorghum  and other grassgenerato C.africana .WorknehandRush(2006)considered thattheuseofconidialconcentrationsof10 5 and10 6 conidia/mLintheirpredictive modelmayresultinanoverestimationofergot severity.Thisapparentdisagreementwithourresultsmaybeduetothenatureoftheexperiments;ourexperimentswereconducted under controlled conditions, while those of Workneh and Rush(2006) were conducted under   󿬁 eld conditions in Texas, whereother abiotic and biotic factors may have in 󿬂 uenced the results.The study reported in this paper has demonstrated that freewater on the sorghum  󿬂 ower stigma aids infection by conidia of  C. africana , and that the infection levels increased as stigmawetness increased. The high infection level (50%) achieved after 4 h of stigma wetness supports the  in vitro  germination  󿬁 ndingsreported by Bhuiyan (2002) and Bhuiyan  et al  . (2002); wheremacro and secondary conidia started to germinate within 4 h of incubation. It has been reported that wet and cloudy weather  predisposes sorghum  󿬂 orets to ergot disease by affecting pollen production and deposition (Quinby 1958). It is likely that onmornings when there is an extended period of dew, infection, particularly of male sterile lines could occur.The present study suggests that conidia germination onstigmas, hence infection, is also affected by a period of stigmawetness. This study also suggests that sorghum spikelets can become infected when the RH is less than 100%. The relativelyhigh infection (11 and 15%) levels which occurred when water was dried immediately after inoculation may be due to the highRH (60  –  100%) in the cabinet after inoculation. Sangitrao and Bade (1979) reported that when humidity is high, rainfall is not essential for ergot development. The importance of high RH(70  –  100%) in sorghum ergot infection has also been reported byother authors (Kulkarni 1942; Futrell and Webster 1966; Molefe1975; Anahosur and Patil 1982).Themethodofdryingoftheinoculatedstigmasmayalsohaveadverselyin 󿬂 uencedtheresults,bykillingsomeoftheconidia,or  Duration of high relative humidity (h) –606121824303642485460667278    %    i  n   f  e  c   t  e   d  s  p   i   k  e   l  e   t  s   /  p  a  n   i  c   l  e 020406080100 Fig. 2.  Infection of sorghum spikelets after various periods of high relativehumidityat20  1  Cinagrowthcabinet.Eachdatapointrepresentsthemean percentageof infectedspikeletsinfourpanicles.Theline representsthe 󿬁 tted values of the data series. Error bars denote the standard error of the means. Duration of high relative humidity (h) 0123456 7    %    i  n   f  e  c   t  e   d  s  p   i   k  e   l  e   t  s   /  p  a  n   i  c   l  e 010203040506070 Fig. 3.  Infection of sorghum spikelets after various periods of high relativehumidityat20  1  Cinagrowthcabinet.Eachdatapointrepresentsthemean% of infected spikelets in four panicles. The lines represent the  󿬁 tted valuesof the data series. Error bars denote the standard error of the means. 498  Australasian Plant Pathology  S. A. Bhuiyan  et al.   perhaps by insuf  󿬁 cient drying. The major source of inoculum of  C. africana  is considered to be airborne secondary conidia(Bandyopadhyay  et al  . 1998), so inoculation methods, such asthat used by Tonapi  et al  . (2003), which do not require the use of aqueous conidial suspensions, may be more appropriate for infection studies. References Anahosur KH, Patil HS (1982) Effect of date of sowing on the incidenceof ergot of sorghum.  Indian Phytopathology  35 , 507  –  509.Bandyopadhyay R, Frederickson DE, McLarenNW, Odvody GN,Ryley MJ(1998) Ergot: a new disease threat to sorghum in the Americas and Australia.  Plant Disease 82 , 356  –  367.doi: 10.1094/PDIS.1998.82.4.356Bhuiyan SA (2002) The biology and control of ergot ( Claviceps africana ) inSorghum. PhD Thesis, The University of Queensland, Gatton.BhuiyanSA,GaleaVJ,RyleyMJ,TayD,LisleAT(2002)Factorsin 󿬂 uencingthe germination of macroconidia and secondary conidia of   Clavicepsafricana. 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(Iowa State University Press:Ames, IA)Sangitrao CS, Bade GH (1979) Meteorological factors associated withhoneydew development and sclerotial stage in sorghum ergot. Sorghum Newsletter   22 , 107  –  108.Tegegne G, Bandyopadhyay R, Mulati T, Kebede Y (1994) Screening for ergot resistance in sorghum.  Plant Disease  78 , 873  –  876.Thakur RP, Rao VP, King SB (1991) In 󿬂 uence of temperature and wetnessduration on infection of pearl millet by  Claviceps fusiformis. Phytopathology  81 , 835  –  838. doi: 10.1094/Phyto-81-835Tonapi V, Ryley M, Galea G, Bhuiyan S, Wearing A (2002) In 󿬂 uence of temperature and relative humidity on pollen traits and ergot severity insorghum.  International Sorghum and Millets Newsletter   43 , 74  –  76.Tonapi VA, Ryley MJ, Galea V, Bhuiyan S, Wearing A (2003) Simpletechniques for production of secondary conidia and ergot inoculation insorghum.  International Sorghum and Millets Newsletter   44 , 97  –  99.Workneh F, Rush CM (2006) Weather factors associated with development of sorghum ergot in the Texas panhandle.  Plant Disease  90 , 717  –  722.doi: 10.1094/PD-90-0717Workneh F, Narasimhan B, Srinivisan R, Rush CM (2006) Assessment of regionalsite-speci 󿬁 csorghumergotseveritypotentialusingradar-rainfallmeasurement.  Plant Disease  90 , 704  –  707. doi: 10.1094/PD-90-0704Manuscript received 2 April 2008, accepted 16 May 2009 Conidial concentration, relative humidity and ergot infection  Australasian Plant Pathology  499http://www.publish.csiro.au/journals/app
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