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Effects of Simulated Rain on the

Persistence of Beauveria bassiana
Conidia on Leaves of Alfalfa and
Wheat
G. D. INGLIS, D. L. JOHNSON & M. S. GOETTEL
Version of record first published: 28 Jun 2010.

To cite this article: G. D. INGLIS, D. L. JOHNSON & M. S. GOETTEL (1995): Effects of

Simulated Rain on the Persistence of Beauveria bassiana Conidia on Leaves of Alfalfa and
Wheat, Biocontrol Science and Technology, 5:3, 365-370

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 Biocontrol Science and Technology (1995) 5, 365± 369

Effects of Simulated Rain on the Persistence of Beauveria

bassiana Conidia on Leaves of Alfalfa and Wheat

G. D. INGLIS,1,2 D. L. JOHNSON 1 AND M . S. GOETTEL 1

1
Agriculture and Agri-Food Canada , Research Centre, PO Box 3000,
Lethbridge, Alberta, Canada T1J 4B1; 2 Centre for Pest Management, Simon
Fraser University, Burnaby, British Columbia, Canada V5A 1S6
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(Received for publication 3 October 1994; revised manuscript accepted 30 April 1995)

The effect of sim ulate d rain at two intensiti es (26.7 or 112.7 mm h 2 1 ) and duratio ns (30 or
60 min) on the persiste nce of Beauveri a bassian a conidi a applie d in water on leave s of alfalfa
leave s of alfalfa (M edicag o sativ a) or wheat (Triticum aestivu m) was investig ated. Initia l
populati ons of conidi a on the leaves of both crops were sim ilar, and ranged from 1.7 3 10 5 to
3.5 3 10 5 colony-f orm ing units cm 2 2 . The sim ulate d rain reduce d the concentratio n of conidi a
on leaves by 28± 61% . A lthoug h there was a sligh t effect due to rain intensit y for alfalfa , there
was no in¯ uenc e of either rain duratio n or crop type on the retentio n of B. bassiana conidia .

Keyw ord s: precipit ation, irrigati on, Beauveri a bassian a, ento mopathog en, epigea l habitats ,
persiste nce

INTR OD UCT ION

The contro l of a variety of insects by Beauveri a bassian a can be obtaine d by the applicati on of
conidi a in epigea l habitat s (Feng et al., 1994). H owever, the persisten ce of conidi a in these
habitat s is usually poor, thereb y reducin g ef® cacy . In additio n to the rapid deactiva tion of conidi a
by ultraviol et radiatio n (Inglis et al., 1995), post-ap plicatio n precipit ation is presu m ed to reduce
conidia l persiste nce (e.g. G ardner et al., 1977 ; Daous t & Pereira , 1986 ; Johnso n et al., 1992).
However, in a recen t ® eld experi m ent, rain appeare d to have little impact on the loss of
B. bassiana conidi a from leave s (Inglis et al., 1993). The objectiv e of this stud y was to verify
® eld observat ions by quantif ying the effec t of rain on the loss of B. bassiana conidi a from leaves
under controll ed conditio ns.

M ATER IALS AN D M ETH OD S

Inoculum Preparatio n and Applicatio n
Dry conidi a of B . bassiana (GH A), supplie d by M ycotec h Corp., Butte , M T , USA , were
suspend ed in sterile deionize d w ater at a concentr ation of 1.7 3 10 9 viable conidi a m l 2 1 . To
facilitat e the dispersi on of conidia , the suspens ions w ere vigorou sly agitate d w ith a micropest le.
The conidi a w ere then applie d to the leave s of w heat plants (Triticum aestivu m, cv. AC R eed)
and alfalfa (Medicag o sativa, cv. AC B lue J), grown individu ally in 8.75-c m diameter pots

0958±3157/95/030365±05 Ó 1995 Crown Coptright

 366 G . D . ING LIS E T A L .

containi ng C ornell mix. Im m ediatel y before treatm ent, the plants w ere trimm ed to a heigh t of
8±10 cm . The conidia l suspens ion (100 m 1) was applie d to individu al plants in a 10.8-c m
diam eter 3 35.5-c m high Plexiglas s cylinde r using an airbrus h (Artek , Rockford , IL, USA ) at
103 kPa. The drople t size, densit y and distribu tion pattern w ere m onitore d usin g water-sen sitiv e
paper (Teeje t Sprayin g System s Co., W heaton , IL, USA ). Follow ing inoculat ion, plants were
maintaine d in the dark at am bien t temperatur e for 15±20 min to allow the water carrie r to
evapora te, before exposin g dry conidi a on leaf surface s to sim ulate d rain.

Rain Sim ulatio n

A continuo us spray , G uelp h rainfal l simulato r (Tossell et al., 1987 ) was used in a glasshou se
with diffuse light and a temperatur e of 23±25 °C . The boo m of the sim ulator w as situate d 1 m
above the bench , and plants w ere rando m ly arrange d within the 0.5 3 0.5-m spra y area; the rain
coverag e within this area had previou sly been determined to be unifor m . Rain w as applie d
(100 kPa) at mediu m (27 m m h 2 1 ) and high (113 m m h 2 1 ) intensiti es using the 1/8 4.3 W and
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1/4 14 W nozzle s respecti vely . Plants were expose d to rain for 30 or 60 m in at each rain level.

Conidial Enum eratio n

Prior to rain exposur e, 10 alfalfa lea¯ ets (ca. 1 3 0.5 cm ) and 10 3 1-cm w heat leaf segm ents
were rando mly collecte d from plants in each pot. The w heat leaf segments w ere rem oved 1±2 cm
from the tip of the lam ina. After exposur e to rain, 10 lea¯ ets or leaf segm ents w ere removed from
the sam e leave s sam pled prio r to rain exposur e. C ontro l treatments w ere alfalfa and w heat plants
inoculat ed w ith conidi a but not exposed , althoug h position ed adjacen t to the rainfal l sim ulator in
the glassho use for 60 m in. No B. bassiana conidi a w ere recover ed from leave s of the alfalf a and
wheat plants sam pled prio r to inoculat ion.
C onidia were recovere d by placin g the leaf samples in 20 m l-vials and washin g them in 5 m l
of 0.01- M phospha te buffe r am ended w ith 0.05 % Tw een-80 (v/v) (buffer ±Tw een) for 2 h on a
rotary shake r (300 rpm ). The w ash solution s w ere diluted three tim es, and a 100 - m l aliquo t from
each dilutio n was spread on a sem i-selecti ve oatm eal±dodin e mediu m (Chase et al., 1986). The
num ber of colony- forming units (CFU ) at the dilutio n yieldin g 30±300 CFU/dish was recorde d
afte r 5±6 days at 25 6 1 °C . The tota l area of the leaf piece s w as determ ined afte r w ashin g with
a leaf area meter (M odel 3100 , Li-Cor Inc., Lincoln , NE, USA ). Leaf areas range d from 4.0 to
11.9 and 4.2 to 10.3 cm 2 for alfalfa and wheat respecti vely . The reductio ns in conidia l number
due to rain exposur e at tim e x w ere calculat ed as: ([CFU cm 2 2 at tim e T 0 2 CFU cm 2 2 at
T x ]/CFU cm 2 2 at T 0) 3 100 . If the num ber of conidi a recover ed at T x was large r than at T 0 , the
percenta ge increas e in number w as calculat ed as: ([C FU cm 2 2 at tim e T 0 2 CFU cm 2 2 at
T x]/C FU cm 2 2 at T x) 3 100.
To assess the effectiv enes s of the wash procedu re, conidi a spraye d on alfalfa and w heat leaves
were recovere d and populat ions cm 2 2 of leaf w ere calculat ed as above. A fter washing , the leaf
segm ents were rinsed in sterile buffer ±Tw een. T hen, 4.5-m m diameter disk s w ere cut from each ,
macerate d, diluted in buffer ±Tw een and the hom ogenat e spread on oatmeal±dodin e agar. The
num ber of C FU cm 2 2 from the washes and hom ogenate s were com pared .

Statistica l Analyses
Each of the three trials conduct ed w as arrange d as a com pletely rando mized desig n with tw o
levels of crop , rain intensit y and duration , each replicate d three tim es. The normality of the
conidia l populat ion data was teste d usin g the Shapiro- W ilk’ s W test. Log 10 transfor mation s were
require d to norm alize the conidia l populati on data , but untrans form ed m eans and standar d errors
of the means ( 6 ) are present ed through out the text. Prior to poolin g the data , the hom ogeneit y
of varianc e between trials w as examined usin g Bartlett ’ s test. The combined data was analyze d
with weighte d analysi s of covarian ce, usin g the genera l linea r m odel routin e of SAS (SA S
Institute , 1988). Conidia l populati ons on leaves before rain exposur e w ere used as the covariat e,
and, in all instance s a trial effect was include d in the m odels. In the absenc e of interacti ons
between crop , rain intensit y, rain duratio n and the covariat e, individu al treatm ents within each
 EF FEC T OF R AIN ON B . BASSIAN A 367

crop w ere compared w ith the contro l treatment usin g weighte d analysi s of covaria nce and
least-squ are means.

RESU LTS A ND D ISCU SSIO N

The wash techniqu e em ployed recovere d 95.6±99.7 % of the conidi a from alfalfa and w heat
leaves . W ithin 10 m in of applicat ion the w ater carrie r had evaporat ed, and initia l populat ions of
dry conidi a on leaves of the plants ranged from 1.6 3 10 5 6 0.39 3 10 5 to
3.5 3 10 5 6 0.64 3 10 5 CFU cm 2 2 (Table 1). There w as no differen ce (P 5 0.28) in the depositi on
of conidi a on the two crops. C onidia of B. bassian a are rapidly killed by solar radiatio n under
® eld conditi ons (Inglis et al., 1993). H ow ever, unde r the diffuse ligh t conditio ns of the
greenho use, the conidi a w ere not affecte d by the 60-m in exposur e (Table 1, contro l treatment) .
The role of precipit atio n on the persiste nce of fungal taxa possessi ng hydroph obic propagu les
(i.e. Beauveria , M etarhizi um and N om uraea ) in epigea l habitat s has not previou sly been studied .
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In this study , it was observe d that sim ulate d rain caused the removal of B. bassiana conidia from
leave s of alfalfa (P 5 0.011) and wheat (P 5 0.007) , w ith the decreas e in populati on densit y
(P < 0.05 ) rangin g from 28.1 6 12.5 to 60.6 6 9.0% (Table 1). The duratio n of rain exposur e had
no effect (P 5 0.42), and conidi a were rem oved equall y from leave s of both crop s (P 5 0.65).
There w as a weak effect (P 5 0.031) due to the differen ce in rain intensit y. A lthoug h the
interacti on betw een crop and rain intensit y was non-sign i® cant (P 5 0.094) , the rain intensit y
effec t occurre d only for alfalfa (T able 1). Rain has been show n to decreas e the folia r persiste nce
of Bacillus thuringi ensis (Frankenh uyzen & Nystro m , 1989), and to facilitat e the dispersa l of
some funga l propagu les (Fitt et al., 1989). D rople t velocit y and size affec t the dispers al of funga l
propagu les (Fitt et al., 1989). A lthoug h the nozzle s and pressur e used produce d a relativel y wide
rang e of drop sizes, the 1-m boo m heigh t selecte d produce d rain w ith relativel y low drople t
velocitie s.
R ain, particula rly that fallin g imm ediatel y afte r the applicat ion of inoculu m, has been
implicate d with decreas ed ef® cacy of B. bassiana by m any research ers (e.g . G ardner et al., 1977;
Johnson et al., 1992). R esults from the presen t stud y indicat e that rain could reduc e the ef® cacy
of this ento m opathog en in epigea l habitats . However, the author s had previou sly observe d that
rain acco m panie d by w ind in excess of 80 km h 2 1 w ithin 30 m in of applicat ion had no apparen t
effec t on persiste nce of B. bassiana conidi a on leaves (Inglis et al., 1993). It now appear s that
the rapid deactiva tion of conidi a by solar radiatio n that occurs unde r ® eld conditio ns obscure s the
effec t of conidia l removal by rain, as determ ined by change s in populati ons over tim e.
A lthoug h simulate d rain rem oves B. bassian a conidi a from leaves , a substant ial proporti on of
the conidi a applie d in water rem ain after exposur e to relative ly high-int ensity rain . Further more,
the effects of rain are not all detrimental, becaus e infectio n can be enhance d unde r conditio ns of
high ambient hum idity (Schae rffenber g, 1964) provide d by precipit atio n or irrigatio n (C am pbell
et al., 1985). W hether precipit atio n w ill enhanc e or jeopardi ze the develop m ent of an epizooti c
will depen d on whethe r conidia l populati ons are decreas ed belo w the inoculu m threshol d
require d to incite mycosis in the targe t insect. Formulatio n adjuvan ts such as stickin g agents
might also reduc e the im pact of rain on conidia l rem oval and prolon g the tim e that inoculu m
densitie s rem ain abov e the threshol d level.

AC KNO W LED GEM E NTS

The author s wish to than k the follo wing people at A gricultur e and Agri-Food Canada, Leth-
bridge : R on Feniuk for his technic al assistanc e, Dr J. J. M iller for the use of the rain sim ulator ,
Toby Entz for his statistica l advice and Drs J. R . Byers, D . A . G audet and L. M . Kawchuk for
their critica l revie ws. The researc h w as funde d in part by a gran t from Farming for the Future ,
Alberta Agricultu ral Researc h Institut e and a Pest M anage m ent A lternativ es Of® ce graduat e
studen t scholars hip to G . D . Inglis. T his manuscri pt is Lethbridg e Researc h Centre contribu tion
#387946 1.
 Downloaded by [University of Western Cape] at 21:25 20 November 2012
368

TABLE 1. Effect of simulated rain on retention of B. bassiana conidia on leaves of alfalfa and wheat

Alfalfa Wheat
a a
Rain Before After Reduction Before After Reduction
2 2 5 2 2 5 2 2 5 2 2 5
treatment (CFU cm 3 10 ) (CFU cm 3 10 ) (% ) (CFU cm 3 10 ) (CFU cm 3 10 ) (% )
b
Medium intensity
c c
30 min 1.96 6 0.35 1.13 6 0.12ab 28.1 6 12.5 1.90 6 0.28 1.10 6 0.20bc 35.4 6 11.80
60 min 2.50 6 0.43 2.09 6 0.52bc 24.6 6 14.0 1 83 6 0.20 0.85 6 0.21ab 56.0 6 6.70
d
High intensity
30 min 2.19 6 0.45 0.74 6 0.24a 60.6 6 9.00 1.60 6 0.39 1.07 6 0.50ab 49.7 6 14.0
60 min 2.58 6 0.81 1.21 6 0.41ab 46.8 6 12.9 1.72 6 0.48 1.16 6 0.55ab 50.7 6 9.70
e
Control 3.51 6 0.64 4.24 6 0.96c 2 2.2 6 16.8 3.07 6 0.46 3.28 6 0.52c 2 4.0 6 7.80
a 2 2 2
G . D . ING LIS E T A L .

Percentage reductions in conidial populations were calculated as: ([CFU cm 2 at time T 0 2 CFU cm 2 at T x ]/CFU cm 2 at T 0 ) 3 100. Percentage increases
2 2 2 2 2 2
were calculated as: ([CFU cm at time T 0 2 CFU cm at T x ]/CFU cm at T x ) 3 100.
b 2 1
Medium-intensity rain 5 26.7 mm h .
c
Post-rain exposure treatment means ( 6 standard error) within each crop not followed by the same letter are signi® cantly different (P < 0.05) according
to least-square means. Conidial populations on leaves after rain exposure were adjusted for the covariate (conidial populations before rain exposure) but
unadjusted means are presented.
d 1
High-intensity rain 5 112.7 mm h 2 .
e
No rain treatment and exposure to the same light conditions for 60 min.
 EF FEC T OF R AIN ON B . BASSIAN A 369

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