Production of Beauveria bassiana fungal spores on rice to control ...
Production of Beauveria bassiana fungal spores on rice to control ...
Production of Beauveria bassiana fungal spores on rice to control ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
<str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>fungal</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> <strong>on</strong> <strong>rice</strong> <strong>to</strong><br />
c<strong>on</strong>trol the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, Hypothenemus hampei, in<br />
Colombia<br />
Francisco J. Posada-Flórez<br />
Centro Naci<strong>on</strong>al de Investigaci<strong>on</strong>es de Café (Cenicafé). A. A. 2427 Manizales. Caldas, Colombia<br />
Abstract<br />
Two isolates <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>fungal</str<strong>on</strong>g> en<strong>to</strong>mopathogen <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) were<br />
grown <strong>on</strong> cooked <strong>rice</strong> using diphasic liquid-solid fermentati<strong>on</strong> in plastic bags <strong>to</strong> produce and harvest spore powder. The<br />
cultures were dried and significant differences were found for isolates and time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest. The <str<strong>on</strong>g>spores</str<strong>on</strong>g> were harvested<br />
manually and mechanically and after the cultures were dried for nine days, when moisture c<strong>on</strong>tent was near 10%. After<br />
harvesting, <str<strong>on</strong>g>spores</str<strong>on</strong>g> were submitted <strong>to</strong> quality c<strong>on</strong>trol <strong>to</strong> assess c<strong>on</strong>centrati<strong>on</strong>, germinati<strong>on</strong>, purity, moisture c<strong>on</strong>tent,<br />
particle size and pathogenicity <strong>to</strong> the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculi<strong>on</strong>idae). Spore<br />
productivity <strong>on</strong> cooked <strong>rice</strong> was less than 1x10 10 <str<strong>on</strong>g>spores</str<strong>on</strong>g>/g using both manually and mechanically harvesting<br />
methodologies. Germinati<strong>on</strong> at 24 hours was over 75% and pathogenicity against H. hampei was over 92.5%. This<br />
methodology is suitable for labora<strong>to</strong>ry and field studies, but not for industrial producti<strong>on</strong> when a high c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> are required for formulati<strong>on</strong> and field applicati<strong>on</strong>s.<br />
Resumen<br />
Dos aislamien<strong>to</strong>s del h<strong>on</strong>go en<strong>to</strong>mopatógeno <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae)<br />
se cultivar<strong>on</strong> en arroz cocido usando el mé<strong>to</strong>do de fermentación difásico líquido – sólido en bolsas plásticas para<br />
producir y cosechar las esporas. Los cultivos se secar<strong>on</strong> y se enc<strong>on</strong>trar<strong>on</strong> diferencias significativas para los aislamien<strong>to</strong>s<br />
y el tiempo de cosecha. Las esporas se cosechar<strong>on</strong> manual y mecánicamente después de secar los cultivos por nueve<br />
días, cuando el c<strong>on</strong>tenido de humedad estuvo cerca del 10%. Después de la cosecha de las esporas, éstas se sometier<strong>on</strong><br />
a c<strong>on</strong>trol de calidad d<strong>on</strong>de se evaluó c<strong>on</strong>centración, germinación, pureza, c<strong>on</strong>tenido de humedad, tamaño de partícula y<br />
pa<strong>to</strong>genicidad sobre la broca del café, Hypothenemus hampei (Ferrari) (Coleoptera: Curculi<strong>on</strong>idae). La producción de<br />
esporas en cultivos de arroz cocido presentó valores menores de 1x10 10 esporas/g para ambas me<strong>to</strong>dologías de cosecha.<br />
La germinación presentó valores superiores a 75% a las 24 horas y la pa<strong>to</strong>genicidad estuvo sobre 92.5%. Esta<br />
me<strong>to</strong>dología de producción del h<strong>on</strong>go B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> es adecuada para estudios de labora<strong>to</strong>rio y campo, pero no para la<br />
producción industrial d<strong>on</strong>de se requiere alta c<strong>on</strong>centración de esporas para la formulación y la aplicación.<br />
Keywords: spore harvesting, germinati<strong>on</strong>, pathogenicity, broca<br />
Palabras claves: cosecha de esporas, germinación, pa<strong>to</strong>genicidad, broca<br />
Corresp<strong>on</strong>dence: fjavierposada@hotmail.com<br />
Received: 30 September 2005 | Accepted: 28 August 2007 | Published: 21 March 2008<br />
Copyright: This is an open access paper. We use the Creative Comm<strong>on</strong>s Attributi<strong>on</strong> 3.0 license that permits unrestricted use, provided that<br />
the paper is properly attributed.<br />
ISSN: 1536-2442 | Volume 8, Number 41<br />
Cite this paper as:<br />
Posada-Flórez FJ. 2008. <str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>fungal</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> <strong>on</strong> <strong>rice</strong> <strong>to</strong> c<strong>on</strong>trol the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, Hypothenemus hampei, in<br />
Colombia. 13pp. Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science 8:41, available <strong>on</strong>line: insectscience.org/8.41<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 1
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Introducti<strong>on</strong><br />
The c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera:<br />
Curculi<strong>on</strong>idae) poses a serious threat <strong>to</strong> c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee<br />
producti<strong>on</strong> throughout the world due <strong>to</strong> its destructi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee seed. Am<strong>on</strong>g the various bioc<strong>on</strong>trol methods<br />
used <strong>to</strong> c<strong>on</strong>trol this insect, the <str<strong>on</strong>g>fungal</str<strong>on</strong>g> en<strong>to</strong>mopathogen<br />
<str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Balsamo) Vuillemin (Hypocreales:<br />
Clavicipitaceae) is highly promising. The fungus has been<br />
shown <strong>to</strong> cause high mortality in various countries where<br />
the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer is present (Fernández et al. 1985;<br />
Lazo 1990; Mendez 1990; Barrios 1992; G<strong>on</strong>zález et al.<br />
1993; Sp<strong>on</strong>agel 1994). However, strains that yield high<br />
spore producti<strong>on</strong>, high pathogenicity and adequate shelf<br />
life remain a challenge for mass producti<strong>on</strong> and field<br />
applicati<strong>on</strong>.<br />
<str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> can be achieved using<br />
different methodologies, which can be classified in<strong>to</strong> low<br />
input and industrial technologies. However, most producti<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>fungal</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> worldwide is carried out using<br />
simple technologies that demand low inputs (Ferr<strong>on</strong><br />
1978; Hussey and Tinsley 1981; Alves and Pereira 1989;<br />
Antía et al. 1992; J<strong>on</strong>es and Burges 1997). Most <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> in Colombia for c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee<br />
berry borer bioc<strong>on</strong>trol is d<strong>on</strong>e using a simple sterilizati<strong>on</strong><br />
technique based <strong>on</strong> cooked <strong>rice</strong> placed inside bottles.<br />
The <str<strong>on</strong>g>spores</str<strong>on</strong>g> are mainly used for field spray applicati<strong>on</strong>s<br />
(Posada 1993; Bustillo and Posada 1996). The <str<strong>on</strong>g>spores</str<strong>on</strong>g> are<br />
harvested by washing them out from the <strong>rice</strong> media with<br />
a 1 % oil-water suspensi<strong>on</strong> (Antía et al. 1992). This<br />
aqueous <str<strong>on</strong>g>spores</str<strong>on</strong>g> suspensi<strong>on</strong> must be used immediately<br />
after preparati<strong>on</strong> <strong>to</strong> avoid spore germinati<strong>on</strong>. Additi<strong>on</strong>ally,<br />
spore l<strong>on</strong>gevity is short if they are kept in the bottles<br />
because high moisture c<strong>on</strong>tent causes rapid loss <str<strong>on</strong>g>of</str<strong>on</strong>g> spore<br />
viability. Harvesting the <str<strong>on</strong>g>spores</str<strong>on</strong>g> from the bottles is time<br />
c<strong>on</strong>suming and if the cultures produced in the bottles are<br />
going <strong>to</strong> be used <strong>to</strong> produce a spore powder, and drying<br />
the <strong>rice</strong> with <str<strong>on</strong>g>spores</str<strong>on</strong>g> is difficult (Posada 1993; G<strong>on</strong>zález et<br />
al. 1993).<br />
Another methodology for spore producti<strong>on</strong> involves the<br />
use <str<strong>on</strong>g>of</str<strong>on</strong>g> fermenters and artificial media. The advantages <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
this technology are that <str<strong>on</strong>g>spores</str<strong>on</strong>g> are easily harvested and<br />
can be used <strong>to</strong> prepare formulati<strong>on</strong>s. In Colombia, some<br />
private companies have been trying <strong>to</strong> develop B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
formulati<strong>on</strong>s as wettable powders and dispersible granules<br />
(Morales and Knauf 1994; Marín et al. 2000). The<br />
quality c<strong>on</strong>trols over those formulati<strong>on</strong>s are not very c<strong>on</strong>sistent,<br />
based <strong>on</strong> tests c<strong>on</strong>ducted by CENICAFÉ, Colombia’s<br />
Nati<strong>on</strong>al C<str<strong>on</strong>g>of</str<strong>on</strong>g>fee Research Centre. Any B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
product that is going <strong>to</strong> be used for the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee growers <strong>to</strong><br />
apply against H. hampei is c<strong>on</strong>tinuously evaluated for<br />
quality, using CENICAFÉ approved methodology (Vélez<br />
et al. 1997; Marín et al. 2000).<br />
Jenkins (1995) reviewed spore yields for 13 fungi produced<br />
<strong>on</strong> semi-solid media and found <strong>on</strong>ly <strong>on</strong>e product<br />
that yielded c<strong>on</strong>centrati<strong>on</strong>s per gram higher than 1 x<br />
10 10 <str<strong>on</strong>g>spores</str<strong>on</strong>g>. The product, based <strong>on</strong> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> that is sold<br />
by Laverlam Internati<strong>on</strong>al corporati<strong>on</strong> www.laverlamintl.com<br />
(previously known as Mycotech) was reported<br />
<strong>to</strong> have 5.8 x 10 10 <str<strong>on</strong>g>spores</str<strong>on</strong>g> per gram <str<strong>on</strong>g>of</str<strong>on</strong>g> substrate. This<br />
implies that the c<strong>on</strong>versi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> substrate <strong>to</strong> <str<strong>on</strong>g>spores</str<strong>on</strong>g> was<br />
high, and that at least some formulati<strong>on</strong>s already in the<br />
market should be able <strong>to</strong> increase yield while remaining<br />
ec<strong>on</strong>omically viable <strong>to</strong> produce.<br />
The development <str<strong>on</strong>g>of</str<strong>on</strong>g> mycoinsecticides <strong>to</strong> be sprayed with<br />
c<strong>on</strong>trolled droplet applicati<strong>on</strong> technology must c<strong>on</strong>sider<br />
the producti<strong>on</strong> and formulati<strong>on</strong> system. The <str<strong>on</strong>g>spores</str<strong>on</strong>g> produced<br />
for oil formulati<strong>on</strong> must be lipophilic, this means<br />
that they should be produced using surface liquid media,<br />
<strong>on</strong> solid substrates or a combinati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> these methodologies<br />
that are called diphasic liquid–solid fermentati<strong>on</strong><br />
(Jenkins and Goettel 1997; Lomer et al. 1997). The<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> that are produced <strong>on</strong> the media after it has dried<br />
can be harvested as a dry powder. Harvesting using fine<br />
sieve meshes prevents clumped <str<strong>on</strong>g>spores</str<strong>on</strong>g> passing through<br />
that can block nozzles and cause difficulties in obtaining<br />
an even distributi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>spores</str<strong>on</strong>g> in the droplets. Also,<br />
harvesting with sieves allows the user <strong>to</strong> know the particle<br />
size, which is an important parameter in quality c<strong>on</strong>trol.<br />
The aim <str<strong>on</strong>g>of</str<strong>on</strong>g> this study was <strong>to</strong> produce B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
using the diphasic liquid–solid fermentati<strong>on</strong> technique<br />
developed for the LUBILOSA (Lutte Biologique c<strong>on</strong>tre<br />
les Locustes et Sauteriaux, www.lubilosa.org) project <strong>to</strong><br />
produce Metarhizium flavoviride (Lomer et al. 1997). The<br />
producti<strong>on</strong> was carried out using two B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> isolates<br />
that showed high virulence <strong>to</strong> the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer. All<br />
the producti<strong>on</strong> steps were closely followed <strong>to</strong> record the<br />
variables related <strong>to</strong> time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest, substrate moisture<br />
c<strong>on</strong>tent, method <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest, particle size and quality c<strong>on</strong>trol<br />
parameters such as: c<strong>on</strong>centrati<strong>on</strong>, germinati<strong>on</strong>,<br />
pathogenicity, powder <str<strong>on</strong>g>spores</str<strong>on</strong>g> moisture c<strong>on</strong>tent and purity.<br />
The <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested were used in subsequent formulati<strong>on</strong><br />
and field applicati<strong>on</strong> experiments.<br />
Materials and Methods<br />
Isolates<br />
Two isolates <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> were prepared: (1) B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
9002 was isolated from the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer H. hampei<br />
(Coleoptera: Curculi<strong>on</strong>idae) collected in Ancuya (Nariño)<br />
in a c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee plantati<strong>on</strong> and (2) B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9205 was isolated<br />
from the sugar cane borer, Diatraea saccharalis (Fabricius)<br />
(Lepidoptera: Pyralidae), collected in Palmira (Valle del<br />
Cauca, Colombia) in a sugar cane plantati<strong>on</strong>. Both isolates<br />
were selected as the most virulent c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer<br />
strains in the CENICAFÉ B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> collecti<strong>on</strong>.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 2
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
<str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g><br />
Five trials were carried out, each <strong>on</strong>e with a batch <str<strong>on</strong>g>of</str<strong>on</strong>g> 30<br />
kg <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> cultured <strong>on</strong> <strong>rice</strong> using LUBILOSA’s<br />
diphasic liquid–solid fermentati<strong>on</strong> methodology (Jenkins,<br />
1995) using plastic bags. Each bag c<strong>on</strong>tained 200 g <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
cooked <strong>rice</strong> inoculated with B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> inoculum grown<br />
<strong>on</strong> liquid media. After the fungus culture had grown,<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> were harvested as powder and then sieved through<br />
three sets sieves with mesh sizes <str<strong>on</strong>g>of</str<strong>on</strong>g> 18 (1 mm), 35 (500 m),<br />
and 60 (250 m). The spore powder, passing through all<br />
three sieves, was used for subsequent research such as<br />
spore s<strong>to</strong>rage in oil, spore oil formulati<strong>on</strong> and feasibility<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> spraying using c<strong>on</strong>trolled droplet applicati<strong>on</strong><br />
technology.<br />
Experimental design<br />
The experiment was set up as a completely randomized<br />
design with a fac<strong>to</strong>rial arrangement (2 x 4). The treatments<br />
were two isolates and four different times <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest,<br />
15, 25, 35 and 45 days after the inoculati<strong>on</strong> and incubati<strong>on</strong>.<br />
Each treatment combinati<strong>on</strong> was replicated<br />
five times and each replicate c<strong>on</strong>sisted <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>on</strong>e bag <str<strong>on</strong>g>of</str<strong>on</strong>g> 200<br />
g. The data were analyzed using <strong>on</strong>e-way analysis <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
variance (ANOVA: SAS Institute 2003)<br />
Spore harvesting and drying<br />
The <str<strong>on</strong>g>spores</str<strong>on</strong>g> were harvested at four different time intervals,<br />
15, 25, 35 and 45 days after the inoculati<strong>on</strong>, <strong>to</strong> evaluate<br />
if harvest time had any effect up<strong>on</strong> spore yield and<br />
<strong>to</strong> obtain an estimate <str<strong>on</strong>g>of</str<strong>on</strong>g> the optimal harvesting period.<br />
To harvest the <str<strong>on</strong>g>spores</str<strong>on</strong>g> as powder it was necessary <strong>to</strong> dry<br />
the fungus <strong>to</strong> reduce moisture c<strong>on</strong>tent and allow the<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> <strong>to</strong> separate from the <strong>rice</strong> substrata. The <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
harvested following this procedure can be preserved for a<br />
l<strong>on</strong>g time without loss <str<strong>on</strong>g>of</str<strong>on</strong>g> germinative power or pathogenicity<br />
(Bateman 1995).<br />
Treatments were selected at random <strong>to</strong> be dried at each<br />
harvest time. To dry the cultures the plastic bags were<br />
opened in a room with a temperature <str<strong>on</strong>g>of</str<strong>on</strong>g> 15 ± 4 o C and<br />
an average relative humidity <str<strong>on</strong>g>of</str<strong>on</strong>g> 55 ±7 % and allowed <strong>to</strong><br />
air dry. Harvesting was d<strong>on</strong>e both mechanically and<br />
manually for 20 minutes. The mechanical harvest involved<br />
the use <str<strong>on</strong>g>of</str<strong>on</strong>g> a shaker Ro-Tap Sieve Shaker (W.S.<br />
Tyler Inc., www.wstyler.com/) that uses horiz<strong>on</strong>tal circular<br />
moti<strong>on</strong> and vertical taping moti<strong>on</strong> <strong>to</strong> stratify and<br />
screen the particles. The manual harvest c<strong>on</strong>sisted <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
back and forth movements <str<strong>on</strong>g>of</str<strong>on</strong>g> the sieve. The samples<br />
were then put through 3 sieves as described above. The<br />
spore powder that was collected after sieving was<br />
weighed and kept in separate sterile vials for further assessments,<br />
such as moisture c<strong>on</strong>tent and quality assessment<br />
(see below) and s<strong>to</strong>red for later use inside a sealed<br />
silica gel vacuum desicca<strong>to</strong>r and kept in a cool room under<br />
the c<strong>on</strong>diti<strong>on</strong>s described above.<br />
Culture moisture c<strong>on</strong>tent assessment<br />
In order <strong>to</strong> m<strong>on</strong>i<strong>to</strong>r air drying <str<strong>on</strong>g>of</str<strong>on</strong>g> the <strong>rice</strong> cultures, each<br />
sample was weighed every day until the weight was<br />
stable. At the same time, from independent samples that<br />
were kept under the experimental c<strong>on</strong>diti<strong>on</strong>s, sub<br />
samples were taken daily <strong>to</strong> assess the humidity c<strong>on</strong>tent<br />
using oven drying where the sub samples were dried for<br />
24 hours at 105°C (Rao et al. 2006). The aim <str<strong>on</strong>g>of</str<strong>on</strong>g> this procedure<br />
was <strong>to</strong> find out how many days it would take the<br />
fungus <strong>to</strong> be ready <strong>to</strong> harvest and also <strong>to</strong> determine the<br />
moisture c<strong>on</strong>tent <str<strong>on</strong>g>of</str<strong>on</strong>g> the <strong>rice</strong> – fungus culture.<br />
Quality c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
The harvested <str<strong>on</strong>g>spores</str<strong>on</strong>g> were evaluated using a quality c<strong>on</strong>trol<br />
methodology developed at CENICAFÉ which assessed<br />
spore c<strong>on</strong>centrati<strong>on</strong>, germinati<strong>on</strong>, pathogenicity,<br />
purity, moisture c<strong>on</strong>tent and particle size (Vélez et al.<br />
1997).<br />
Table 1. Weight (g) <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture and <strong>rice</strong> at different times <str<strong>on</strong>g>of</str<strong>on</strong>g> drying and before the <str<strong>on</strong>g>spores</str<strong>on</strong>g> were harvested.<br />
Days <str<strong>on</strong>g>of</str<strong>on</strong>g> drying N<br />
Time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest Bb 9002 (days)<br />
Time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest Bb 9205 (days)<br />
15 25 35 45 15 25 35 45<br />
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD<br />
1 10 171.1±4.6 a 170.6±10.6 a 159.2±7.7 a 151.7±8.1 a 174.5±6.5 a 167.3±5.9 a 155.3±11.9 a 151.5±5.7 a<br />
2 10 145±11.3 b 132.3±10.4 b 106.9±6.9 b 86.5±8.8 b 141.3±5.7 b 123.9±6.4 b 109.3±10.6 b 92.0±8.4 b<br />
3 10 120.9±6.3 c 105.8±10 c 85.1±7.1 c 63.7±6.5 c 120.1±6.3 c 99.9±6.8 c 87.7±10.1 c 69.2±8.9 c<br />
4 10 100.7±6.3 d 96.8±9.5 cd 71.3±7.1 d 55.6±4.4 d 103.5±6.9 d 90.1±6.6 d 73.1±9.8 d 55.4±8 d<br />
5 10 90.5±4.4 e 90.9±8.5 ed 66.5±7.5 d 54.1±4.3 d 93.5±6.2 e 81.3±6.2 de 63.4±9.2 de 48.3±5.9 de<br />
6 10 87.4±3.7 e 84.1±9.1 ed 64.8±7.7 d 53.5±3.8 d 87.3±5 ef 74.8±6.3 ef 58.0±8.6 e 45.4±4.2 e<br />
7 10 86.6±3.5 e 80.7±8.4 e 64.3±7.8 d 53.5±3.8 d 84.2±4.2 f 70.2±6.7 f 55.2±7.9 e 44.3±3.4 e<br />
8 10 86.3±3.5 e 80.4±8.3 e 63.9±7.9 d 53.4±3.9 d 82.7±3.8 f 69.5±6.8 f 54.5±7.9 e 44.2±3.3 e<br />
9 10 86.1±3.4 e 80.4±8.3 e 63.9±7.9 d 53.3±3.9 d 81.9±3.8 f 69.5±6.8 f 54.5±7.9 e 44.0 e 3.1<br />
Means followed by the same letter are not significantly different from each other, Tukey (P = 0.05) (SAS, 2003).<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 3
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Figure 1. Weight loss by B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> strains cultured <strong>on</strong> <strong>rice</strong> during oven drying following harvesting <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g>. Bars represent c<strong>on</strong>fidence<br />
intervals for means (P = 0.05).<br />
Results<br />
Moisture c<strong>on</strong>tent lost and dry weight <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture <strong>on</strong> <strong>rice</strong><br />
Table 1 shows the results <str<strong>on</strong>g>of</str<strong>on</strong>g> moisture loss during air drying<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>rice</strong> substrate use <strong>to</strong> culture two B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> isolates<br />
for spore producti<strong>on</strong> throughout four different harvest<br />
times and the final dry weight. Initially, the weight <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
<strong>rice</strong> used as substrate plus B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> liquid inoculum <strong>to</strong><br />
start the fungus culture was 200 g wet weight per plastic<br />
bag and the weight was recorded just prior <strong>to</strong> first<br />
Table 2. Analysis <str<strong>on</strong>g>of</str<strong>on</strong>g> variance for dry weight <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture <strong>on</strong> <strong>rice</strong> <strong>to</strong> harvest the<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g>.<br />
Source <str<strong>on</strong>g>of</str<strong>on</strong>g> variati<strong>on</strong><br />
DF Sums <str<strong>on</strong>g>of</str<strong>on</strong>g> squares Mean squares F value Pr > F<br />
Isolate 1 2987.0. 2987 59.0. 0.0001<br />
Harvest day 3 173110.5 57703.5 1138.9 0.0001<br />
Isolate * harvest day 3 1386 462 9.1 0.0001<br />
Dry day 8 671096.5 83887.1 1655.6 0.0001<br />
Isolate * dry day 8 2102.8 262.9 5.2 0.0001<br />
Harvest day * dry day 24 9533.3 397.2 7.8 0.0001<br />
Isolate * harvest day * dry day 24 1152.3 48 1.0. 0.5358<br />
General linear models procedure (SAS Institute, 2003)<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 4
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
harvest. The daily moisture loss <str<strong>on</strong>g>of</str<strong>on</strong>g> the B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture<br />
and <strong>rice</strong> shows a similar tendency for both isolates and<br />
was directly related <strong>to</strong> the harvest time. The moisture loss<br />
was greater the l<strong>on</strong>ger the time before <str<strong>on</strong>g>spores</str<strong>on</strong>g> were harvested.<br />
The moisture loss fac<strong>to</strong>r at each spore harvest was<br />
determined by doing a divisi<strong>on</strong> between wet and dry cultures.<br />
The values <str<strong>on</strong>g>of</str<strong>on</strong>g> the fac<strong>to</strong>r obtained were 2.4, 2.4, 3.2<br />
and 3.8 at 15, 25, 35 and 45 days respectively. These<br />
data estimate the amount <str<strong>on</strong>g>of</str<strong>on</strong>g> cooked <strong>rice</strong> and dry <strong>rice</strong> that<br />
need <strong>to</strong> be produced and harvested <strong>to</strong> obtain spore<br />
powder. These estimates were necessary so that appropriate<br />
quantities <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> could be produced for field experiments.<br />
At each harvest time the wet <strong>to</strong> dry fac<strong>to</strong>r was<br />
the same for 15 and 25 days and higher for 35 and 45<br />
days. Analysis <str<strong>on</strong>g>of</str<strong>on</strong>g> variance <str<strong>on</strong>g>of</str<strong>on</strong>g> isolate, time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest and<br />
culture drying time were not statistically significant different<br />
for the interacti<strong>on</strong> between fac<strong>to</strong>rs (df = 24, F = 0.95,<br />
P = 0.5358). However, the statistical analysis (ANOVA)<br />
(Table 2) <str<strong>on</strong>g>of</str<strong>on</strong>g> independent fac<strong>to</strong>rs showed significant differences.<br />
The analysis <str<strong>on</strong>g>of</str<strong>on</strong>g> variance <str<strong>on</strong>g>of</str<strong>on</strong>g> isolate and period <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
harvest per day that it <strong>to</strong>ok <strong>to</strong> dry the cultures were significantly<br />
different (df = 81, P = 0.0001). The significant<br />
differences were for isolates and time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest mainly<br />
between the first five days <str<strong>on</strong>g>of</str<strong>on</strong>g> drying cultures.<br />
Moisture c<strong>on</strong>tent assessment <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
culture and <strong>rice</strong> before harvesting the <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
Figure 1 presents the moisture loss <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture<br />
and <strong>rice</strong> during air drying process before the <str<strong>on</strong>g>spores</str<strong>on</strong>g> were<br />
harvested. The B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture and <strong>rice</strong> in <strong>to</strong> each<br />
treatment were dried for nine days at which point the<br />
moisture c<strong>on</strong>tent was stable (Figure 2). The moisture c<strong>on</strong>tent<br />
fell rapidly during the first four days and there was<br />
variati<strong>on</strong> between days for different isolates and times <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
harvest (Table 1, Figure 2). After five days the moisture<br />
loss was relatively small and remained steady at around<br />
10 % until the ninth day. This final moisture c<strong>on</strong>tent was<br />
determined and m<strong>on</strong>i<strong>to</strong>red drying culture and <strong>rice</strong> subsamples<br />
daily in an oven in order <strong>to</strong> harvest the <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
with the lowest moisture c<strong>on</strong>tent.<br />
Quality c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> harvested powder <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
Spore powder weight<br />
Figure 3 shows the spore powder weight harvested from<br />
B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> isolates using manual and mechanical methods<br />
at different times <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest. The analysis <str<strong>on</strong>g>of</str<strong>on</strong>g> variance<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> isolate, method and time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest showed significant<br />
differences for fac<strong>to</strong>r interacti<strong>on</strong>s (df = 3, F = 24.7, P =<br />
0.0001).<br />
Spore powder producti<strong>on</strong> showed c<strong>on</strong>siderable variati<strong>on</strong>.<br />
<str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> both isolates was higher using the manual<br />
method compared with the mechanical method. The<br />
overall spore producti<strong>on</strong> by isolate shows that B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
9205 was slightly more productive than B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002<br />
and the highest producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> both isolates was obtained<br />
by manually harvesting the <str<strong>on</strong>g>spores</str<strong>on</strong>g> 15 days after<br />
inoculati<strong>on</strong>.<br />
Spore powder producti<strong>on</strong>: c<strong>on</strong>centrati<strong>on</strong><br />
Figure 4 shows the spore c<strong>on</strong>centrati<strong>on</strong> produced by<br />
both isolates and harvesting methods estimated as grams<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> spore powder. The spore c<strong>on</strong>centrati<strong>on</strong> was<br />
highly variable for harvest methods and for isolates. The<br />
spore c<strong>on</strong>centrati<strong>on</strong> was higher for the mechanical method.<br />
This result was the opposite for the spore powder<br />
weight where a higher yield was obtained using the<br />
manual method.<br />
Spore c<strong>on</strong>centrati<strong>on</strong> was higher when harvested earlier<br />
compared with later times. It was observed that after harvesting<br />
the <strong>rice</strong> cultures B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> remained <strong>on</strong><br />
the <strong>rice</strong>. Following sieving these cultures were washed out<br />
with water plus 1% <str<strong>on</strong>g>of</str<strong>on</strong>g> emulsifiable oil <strong>to</strong> extract the<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> for counting and <strong>to</strong> calculate the <strong>to</strong>tal c<strong>on</strong>centrati<strong>on</strong><br />
for each treatment.<br />
Table 3 shows by isolate, time and method <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest, the<br />
<strong>to</strong>tal producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> estimated from the c<strong>on</strong>centrati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> spore powder and <str<strong>on</strong>g>spores</str<strong>on</strong>g> remaining in the <strong>rice</strong><br />
after harvest. The extracti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the remaining <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
from the <strong>rice</strong> cultures showed that a high number <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> remain in the <strong>rice</strong> after sieving; the number <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> that remained <strong>on</strong> the <strong>rice</strong> was greater than the<br />
number <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> that were harvested as powder. The<br />
data show that B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9205 is more productive than<br />
B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002. <str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9205 was higher<br />
15 days after inoculati<strong>on</strong> and thereafter declined<br />
gradually <strong>to</strong> 45 days, which resembles a typical growth<br />
microbial curve. In c<strong>on</strong>trast, B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002 did not<br />
show this pattern <str<strong>on</strong>g>of</str<strong>on</strong>g> spore producti<strong>on</strong>.<br />
Although the <strong>to</strong>tal spore producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> isolate B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
9205 was higher than B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002 (Figure 4, Table 3)<br />
the spore powder weight was higher for B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002,<br />
which was not desirable for formulati<strong>on</strong> preparati<strong>on</strong> because<br />
a high proporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the powder was starch.<br />
Germinati<strong>on</strong> test<br />
The germinati<strong>on</strong> test was c<strong>on</strong>ducted at 24 and 48 hours.<br />
The test was d<strong>on</strong>e for all harvest periods and isolates.<br />
Overall, the germinati<strong>on</strong> for all treatments was over 75%<br />
and germinati<strong>on</strong> at 24 hours was over 80%. Germinati<strong>on</strong><br />
counts at 48 hours were difficult <strong>to</strong> perform because they<br />
were overgrown with mycelia and the risk <str<strong>on</strong>g>of</str<strong>on</strong>g> mistakes<br />
was higher. The germinati<strong>on</strong> tested at 24 hours for B.<br />
<str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9205 was higher after 25 days <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest, while B.<br />
<str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002 was higher after 35 and 45 days.<br />
Pathogenicity test<br />
Table 4 shows the pathogenicity results <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> using the CENICAFÉ bioassay methodology<br />
(Vélez et al. 1997). The mortality <str<strong>on</strong>g>of</str<strong>on</strong>g> H. hampei was not<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 5
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Figure 2. Loss <str<strong>on</strong>g>of</str<strong>on</strong>g> moisture c<strong>on</strong>tent during air drying <str<strong>on</strong>g>of</str<strong>on</strong>g> two strains <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> cultured <strong>on</strong> <strong>rice</strong> at four different times previous <strong>to</strong><br />
spore harvest. Bars represent c<strong>on</strong>fidence intervals for means (P = 0.05).<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 6
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Figure 3. Weight <str<strong>on</strong>g>of</str<strong>on</strong>g> spore powder harvested from two strains <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> cultured <strong>on</strong> <strong>rice</strong> using two methods <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest at four<br />
different times. Bars represent c<strong>on</strong>fidence intervals for means (P= 0.05).<br />
significantly different between isolates and days <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest<br />
(df = 3, F = 1.5, P = 0.2089) and was over 92 % for both<br />
isolates and for all different times <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested.<br />
The mean mortality time (MMT in Table 4) in days <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
H. hampei treated with the <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested in this experiment<br />
was not significantly different between isolates and<br />
days <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest (df = 3, F = 0.97, P = 0.4077). Isolate B.<br />
<str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002 at harvest times <str<strong>on</strong>g>of</str<strong>on</strong>g> 15, 25 and 45 days did<br />
not show a significant difference in mean mortality time,<br />
but between 15 days and the <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested 35 days<br />
after inoculati<strong>on</strong> there was a significant difference (df =<br />
156, F = 4.4, P = 0.0056). Isolate B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9205 harvested<br />
at 15 and 25 days showed significant differences in<br />
mean mortality time compared with the <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested<br />
at 35 and 45 days. For both isolates, <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested at<br />
15 days were more pathogenic <strong>to</strong> H. hampei than <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
harvested after 35 days. The <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested at days 35<br />
and 45 showed no significant differences between them<br />
(df = 156, F = 6.3, P = 0.0004).<br />
Purity<br />
The presence <str<strong>on</strong>g>of</str<strong>on</strong>g> c<strong>on</strong>taminant microorganisms was low,<br />
less than 2 % <str<strong>on</strong>g>of</str<strong>on</strong>g> col<strong>on</strong>y-forming unit for both isolates and<br />
was present <strong>on</strong>ly in the <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested <strong>on</strong> day 25. Only<br />
the presence <str<strong>on</strong>g>of</str<strong>on</strong>g> c<strong>on</strong>taminant fungi was detected. These<br />
were identified as Penicillium spp. and Cladosporium spp.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 7
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Figure 4. Spore producti<strong>on</strong> per gram <str<strong>on</strong>g>of</str<strong>on</strong>g> two isolates <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> cultured <strong>on</strong> <strong>rice</strong> harvested using two methods and four times<br />
after inoculati<strong>on</strong>. Bars represent c<strong>on</strong>fidence intervals for means (P= 0.05).<br />
Spore powder moisture c<strong>on</strong>tent<br />
The moisture c<strong>on</strong>tent <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> spore powder harvested<br />
at different times showed a tendency <strong>to</strong> be lower for<br />
isolate 9002 and was higher for the <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvest after<br />
15 days. At this time 9205 had moisture c<strong>on</strong>tent <str<strong>on</strong>g>of</str<strong>on</strong>g> 13 ±<br />
3.1 % compared <strong>to</strong> 11.4 ± 0.5 % for isolate 9002. After<br />
harvest at 25 and 35 days the moisture c<strong>on</strong>tent was similar<br />
for both isolates. At 45 days the moisture c<strong>on</strong>tent<br />
dropped dramatically and was 6.3 ± 0.1 % for 9205 and<br />
5.9 ± 0.9 % for 9002.<br />
Spore powder particle size<br />
The <str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested were taken from the bot<strong>to</strong>m <str<strong>on</strong>g>of</str<strong>on</strong>g> a set<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> sieves <str<strong>on</strong>g>of</str<strong>on</strong>g> size 18 (1 mm), 35 (500 m) and 60 (250 m);<br />
therefore, the spore powder that was obtained had<br />
particle sizes <str<strong>on</strong>g>of</str<strong>on</strong>g> less than 250 m.<br />
Discussi<strong>on</strong><br />
Moisture loss was a result <str<strong>on</strong>g>of</str<strong>on</strong>g> the metabolic activity <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
fungus, transpirati<strong>on</strong> and diffusi<strong>on</strong> while the cultures<br />
were developing. Moisture was also lost when the cultures<br />
were dried for nine days in a cool room with 55 ± 7<br />
% average relative humidity. The informati<strong>on</strong> obtained<br />
from drying suggests that the cultures can be dried in five<br />
days following the same process as in this study, which<br />
would save time and resources.<br />
The lowest moisture c<strong>on</strong>tent achieved from harvested<br />
spore powder was around 10% for the best harvest period<br />
(15 days). This moisture c<strong>on</strong>tent, obtained under c<strong>on</strong>trolled<br />
c<strong>on</strong>diti<strong>on</strong>s and after nine days <str<strong>on</strong>g>of</str<strong>on</strong>g> air drying was<br />
still <strong>to</strong>o high <strong>to</strong> s<strong>to</strong>re the <str<strong>on</strong>g>spores</str<strong>on</strong>g> and preserve their viability.<br />
Ideally, a moisture c<strong>on</strong>tent <str<strong>on</strong>g>of</str<strong>on</strong>g> 5% would have been<br />
preferable (Bateman 1995). One way <strong>to</strong> decrease the<br />
moisture c<strong>on</strong>tent would be <strong>to</strong> keep the spore mix with a<br />
desiccant such as silica gel which would help <strong>to</strong> achieve<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 8
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Table 3. C<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> two strains <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> harvested as powder from <strong>rice</strong> cultures, <str<strong>on</strong>g>spores</str<strong>on</strong>g> remaining<br />
attached <strong>to</strong> dried <strong>rice</strong> after harvest and <strong>to</strong>tal estimate <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> producti<strong>on</strong> per gram.<br />
Isolate Harvest<br />
days<br />
Spores powder harvested<br />
Mean ± CI * .<br />
Spores remaining <strong>on</strong><br />
<strong>rice</strong> Mean ± CI * .<br />
Manual<br />
Total <str<strong>on</strong>g>spores</str<strong>on</strong>g> producti<strong>on</strong><br />
Mean ± CI * .<br />
Bb9002 15 1.2 x10 9 ± 1.5x10 9 1.3x10 9 ± 2.0x10 8 2.4x10 9 ± 1.6x10 9<br />
Bb9002 25 5.5 x10 8 ± 1.5x10 8 1.1x10 9 ± 4.0x10 8 1.6x10 9 ± 3.3x10 8<br />
Bb9002 35 1.9 x10 9 ± 1.3x10 9 7.1x10 8 ± 1.7x10 8 2.6x10 9 ± 1.4x10 9<br />
Bb9002 45 1.3 x10 9 ± 9.0x10 8 3.6x10 8 ± 9.0x10 7 1.6x10 9 ± 9.2x10 8<br />
Bb9205 15 5.7 x10 9 ± 1.2x10 9 1.6x10 9 ± 2.5x10 8 7.4x10 9 ± 1.3x10 9<br />
Bb9205 25 3.0 x10 9 ± 1.7x10 9 7.7x10 8 ± 1.1x10 8 3.7x10 9 ± 1.7x10 9<br />
Bb9205 35 4.2 x10 9 ± 1.1x10 9 3.1x10 8 ± 1.5x10 8 4.5x10 9 ± 9.4x10 8<br />
Bb9205 45 3.6 x10 9 ± 1.9x10 9 6.1x10 8 ± 1.0x10 8 4.2x10 9 ± 1.9x10 9<br />
Shaker<br />
Bb9002 15 7.2x10 8 ± 4.4x10 8 7.6x10 8 ± 2.1x10 8 1.5x10 9 ± 4.2x10 8<br />
Bb9002 25 1.4x10 9 ± 1.0x10 9 4.9x10 8 ± 1.6x10 8 1.9x10 9 ± 1.1x10 9<br />
Bb9002 35 2.5x10 9 ± 8.0x10 8 1.5x10 9 ± 3.9x10 8 3.9x10 9 ± 1.2x10 9<br />
Bb9002 45 1.8x10 9 ± 4.7x10 8 5.5x10 8 ± 1.7x10 8 2.3x10 9 ± 4.1x10 8<br />
Bb9205 15 7.3x10 9 ± 4.6x10 9 8.5x10 8 ± 2.8x10 8 8.1x10 9 ± 4.7x10 9<br />
Bb9205 25 5.0x10 9 ± 2.2x10 9 2.2x10 7 ± 1.0x10 7 5.0x10 9 ± 2.2x10 9<br />
Bb9205 35 6.6x10 9 ± 1.7x10 9 3.2x10 7 ± 1.7x10 7 6.6x10 9 ± 1.7x10 9<br />
Bb9205 45 3.3x10 9 ± 6.5x10 8 2.4x10 8 ± 2.8x10 8 3.5x10 9 ± 7.8x10 8<br />
* CI c<strong>on</strong>fidence intervals for mean (P= 0.05).<br />
the objective <str<strong>on</strong>g>of</str<strong>on</strong>g> spore s<strong>to</strong>rage with low moisture c<strong>on</strong>tent<br />
(Moore and Caudwell 1997). The use <str<strong>on</strong>g>of</str<strong>on</strong>g> silica gel <strong>to</strong><br />
maintain the spore moisture at a low level would not pose<br />
a technical problem even for larger scale producti<strong>on</strong>.<br />
Moisture c<strong>on</strong>tent can influence the weight <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>spores</str<strong>on</strong>g><br />
harvested. Although the <str<strong>on</strong>g>spores</str<strong>on</strong>g> with the highest weight<br />
also have high moisture c<strong>on</strong>tent, this did not mean that<br />
they have high spore c<strong>on</strong>centrati<strong>on</strong>s due <strong>to</strong> the presence<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> starch from the <strong>rice</strong>. Starch could block the nozzles <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
applicati<strong>on</strong> equipment. These characteristics need <strong>to</strong> be<br />
evaluated if <str<strong>on</strong>g>fungal</str<strong>on</strong>g> producti<strong>on</strong> is <strong>to</strong> be scaled up <strong>to</strong> avoid<br />
applicati<strong>on</strong> problems in the field situati<strong>on</strong>s. The powder<br />
harvested from B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> cultures <strong>on</strong> <strong>rice</strong> is a mixture <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
starch and <str<strong>on</strong>g>spores</str<strong>on</strong>g>. The high weight <str<strong>on</strong>g>of</str<strong>on</strong>g> powder harvested<br />
from B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002, which had low spore c<strong>on</strong>centrati<strong>on</strong>,<br />
means that there is a difference between B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
isolates in the ratio <str<strong>on</strong>g>of</str<strong>on</strong>g> starch <strong>to</strong> <str<strong>on</strong>g>fungal</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> and this<br />
needs <strong>to</strong> be c<strong>on</strong>sidered in mass producti<strong>on</strong>.<br />
The harvest methods used showed differences in the<br />
amount <str<strong>on</strong>g>of</str<strong>on</strong>g> powder extracted from the cultures and the<br />
c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> per gram <str<strong>on</strong>g>of</str<strong>on</strong>g> powder. The possible<br />
reas<strong>on</strong> for this result was that the mechanical<br />
Table 4. Pathogenicity <str<strong>on</strong>g>of</str<strong>on</strong>g> two strains <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> against the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer evaluated with<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> harvested at four different times.<br />
Harvest days after innoculat<strong>on</strong><br />
Bb 9002 Bb 9205<br />
Mortality (%) MMT * (Days) Mortality (%) MMT * (Days)<br />
Mean ± SD Mean ± SD Mean ± SD Mean ± SD<br />
15 100±0 a 3.2±0.4 a 97.5±15.8 a 3.1±1.5 a<br />
25 92.5±26.7 a 3.4 ±1.4 ab 95.0±22.1 a 3.3±0.9 a<br />
35 100±0 a 3.9±0.3 b 97.5±15.8 a 4.0±1.1 b<br />
45 92.±26.75 a 3.6±1.1 ab 100±0 a 4.0±1 b<br />
* MMT = mean mortality time Means followed by the same letter are not significantly different from<br />
each other, Tukey (P = 0.05) (SAS, 2003)<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 9
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> spore producti<strong>on</strong> needs <strong>to</strong> be highly efficient<br />
and productive <strong>to</strong> make a successful, inexpensive mycopesticide<br />
based solely <strong>on</strong> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g>. Current<br />
diphasic liquid solid fermentati<strong>on</strong> technologies are incapable<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> yielding mycoinsecticide c<strong>on</strong>centrati<strong>on</strong>s higher<br />
than 1x10 10 (Jenkins 1995). In additi<strong>on</strong> <strong>to</strong> producti<strong>on</strong><br />
c<strong>on</strong>straints, en<strong>to</strong>mopthogenic fungi have <strong>to</strong> compete<br />
against chemical insecticides and require several applicati<strong>on</strong>s<br />
during the crop cycle (Yendol and Roberts 1970).<br />
They also need <strong>to</strong> be comparable in terms <str<strong>on</strong>g>of</str<strong>on</strong>g> efficacy,<br />
achieving mortality rates higher than 80 % under field<br />
c<strong>on</strong>diti<strong>on</strong>s (Thomas et al. 1997).<br />
The methodology evaluated in this study allows for the<br />
producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> high quality B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g>, but the<br />
quantities produced are <strong>on</strong>ly suitable for small-scale<br />
labora<strong>to</strong>ry and field trials. All the c<strong>on</strong>straints analyzed<br />
above do not imply that the use <str<strong>on</strong>g>of</str<strong>on</strong>g> B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> against the<br />
c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer should be s<strong>to</strong>pped. In additi<strong>on</strong> <strong>to</strong> this<br />
fungus being the most important natural enemy <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer (Ruíz 1996), field studies using B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
and have shown that it is a promising strategy and a<br />
key comp<strong>on</strong>ent <str<strong>on</strong>g>of</str<strong>on</strong>g> the integrated pest management <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer even though they are in their initial<br />
stages (Posada 1998; De la Rosa et al. 2000; Haraprasad<br />
et al. 2001; Posada et al. 2004). The use <str<strong>on</strong>g>of</str<strong>on</strong>g> en<strong>to</strong>mopathogenic<br />
fungi is a technology that is still being developed<br />
and improvements in producti<strong>on</strong>, formulati<strong>on</strong> and field<br />
applicati<strong>on</strong> are needed. There are several alternatives for<br />
using the fungus against the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, such as<br />
“inoculum-introducti<strong>on</strong>” or “augmentative -inoculative<br />
strategies” (Hajek 1993) that can c<strong>on</strong>tribute <strong>to</strong> an epizootic<br />
c<strong>on</strong>diti<strong>on</strong> and create a permanent mortality fac<strong>to</strong>r in<br />
the field.<br />
Acknowledgments<br />
Thanks <strong>to</strong> the British government through ODA, IIBC,<br />
the British Council and <strong>to</strong> the Federación Naci<strong>on</strong>al de<br />
Cafeteros de Colombia through Cenicafé for financing<br />
this research. Thanks <strong>to</strong> M<strong>on</strong>ica Pava- Ripoll, T<strong>on</strong>y<br />
Little, Mari<strong>on</strong> Seier, Valerie Walters, Adrian Leach,<br />
Carlos Quintero, Eduardo Osorio and Patricia Marín for<br />
their help. Fernando Vega and Ann Simpkins provided<br />
helpful review and comments <strong>on</strong> an early versi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
manuscript.<br />
Table 5. Relati<strong>on</strong>ship between time <str<strong>on</strong>g>of</str<strong>on</strong>g> harvest, <str<strong>on</strong>g>spores</str<strong>on</strong>g> produced by <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> 9002 cultured <strong>on</strong> wet <strong>rice</strong>,<br />
spore c<strong>on</strong>centrati<strong>on</strong> and the producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> wet and dry <strong>rice</strong> necessary <strong>to</strong> treat a number <str<strong>on</strong>g>of</str<strong>on</strong>g> c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee trees and hectares.<br />
Harvest<br />
period<br />
(Days after<br />
inoculati<strong>on</strong>)<br />
Spore<br />
powder *<br />
produced<br />
per kg <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
wet <strong>rice</strong> (g)<br />
Spore c<strong>on</strong>centrati<strong>on</strong><br />
in the spore<br />
powder harvested<br />
(<str<strong>on</strong>g>spores</str<strong>on</strong>g>/gram)<br />
Spore producti<strong>on</strong><br />
per<br />
kg <str<strong>on</strong>g>of</str<strong>on</strong>g> wet<br />
<strong>rice</strong><br />
(<str<strong>on</strong>g>spores</str<strong>on</strong>g>)<br />
<str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>rice</strong> <strong>to</strong> treat 5000<br />
c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee trees using (= 1 x 10 10<br />
spore/tree)<br />
Wet producti<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<strong>rice</strong> (kg)<br />
1 ha 1000<br />
ha<br />
Ratio<br />
wet <strong>to</strong><br />
dry 1<br />
ha<br />
Dry producti<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<strong>rice</strong> (kg)<br />
1000<br />
ha<br />
A B C D E F G H I<br />
15 9.5 5.7 x10 10 5.4 x10 11 92.3 92,336 2.4 39.2 39,197<br />
25 9 5.0x10 10 4.5 x 10 11 111.1 111,111 2.4 45.6 45,556<br />
35 8.5 2.0x10 10 1.7 x10 11 294.1 294,118 3.2 92.5 92,500<br />
45 8 3.0x010 10 2.4 x 10 11 208.3 208,333 3.8 55 55,000<br />
* Based <strong>on</strong> 200 g <str<strong>on</strong>g>of</str<strong>on</strong>g> wet <strong>rice</strong> and after Bb cultured <strong>on</strong> <strong>rice</strong> was dried and the spore powder was harvested.<br />
The figures given in the above Table were calculated using the data for Bb 9002 isolate harvested<br />
manually. A = For each period <str<strong>on</strong>g>of</str<strong>on</strong>g> harvested <str<strong>on</strong>g>spores</str<strong>on</strong>g>, spore powder weight was obtained and<br />
the spore c<strong>on</strong>centrati<strong>on</strong> per gram was estimated. B = The mean spore powder harvested at 15 days<br />
was 1.9 g from 200 g <str<strong>on</strong>g>of</str<strong>on</strong>g> wet <strong>rice</strong>, thus the spore powder harvested from 1000 g <str<strong>on</strong>g>of</str<strong>on</strong>g> wet <strong>rice</strong> will be<br />
9.5 g. C = The mean c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> per gram <str<strong>on</strong>g>of</str<strong>on</strong>g> powder harvested at 15 days was 5.7 x<br />
10 10 <str<strong>on</strong>g>spores</str<strong>on</strong>g>/gram. D = B x C: 9.5 g x 5.7 x 10 10 <str<strong>on</strong>g>spores</str<strong>on</strong>g>/g = 5.4 x 10 11 <str<strong>on</strong>g>spores</str<strong>on</strong>g>. E = kg wet <strong>rice</strong> x<br />
<str<strong>on</strong>g>spores</str<strong>on</strong>g> (ha)/<str<strong>on</strong>g>spores</str<strong>on</strong>g> (1 kg wet <strong>rice</strong>): 1 kg x 5 x 10 13 <str<strong>on</strong>g>spores</str<strong>on</strong>g>/5.4 x 10 11 <str<strong>on</strong>g>spores</str<strong>on</strong>g> = 92.3 kg/ha <str<strong>on</strong>g>of</str<strong>on</strong>g> wet <strong>rice</strong>.<br />
F = E x 1000 ha. G = Is the ratio between wet <strong>rice</strong> + B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> culture and dried <strong>rice</strong> + B. <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
culture. H = E/H: 92.3 kg/2.4 = 39.2 kg. I = F/H: 92 336/2.4 = 39 197 kg. The number <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> produced<br />
by 1 kg <str<strong>on</strong>g>of</str<strong>on</strong>g> dry <strong>rice</strong> was established as follows: (dose/ha)/H = 5 x 10 13 /39.2 = 1.3 x 10 12 /2.4<br />
(ratio wet/dry) = 5.4 x 10 11 <str<strong>on</strong>g>spores</str<strong>on</strong>g>/kg <str<strong>on</strong>g>of</str<strong>on</strong>g> dry <strong>rice</strong>. It has the same number <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>spores</str<strong>on</strong>g> as 1 kg <str<strong>on</strong>g>of</str<strong>on</strong>g> wet<br />
<strong>rice</strong>.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 11
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
References<br />
Alves SB, Pereira RM. 1989. <str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> Metarhizium anisoplae<br />
(Metsch.) Sorok. and <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Bals.) Vuill. in plastic trays.<br />
Ecossistema 14: 188-192.<br />
Antía OP, Posada FJ, Bustillo AE, G<strong>on</strong>záles MT. 1992. Producción en<br />
finca del h<strong>on</strong>go <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> para el c<strong>on</strong>trol de la broca del<br />
café. Cenicafé (Colombia). Avances Técnicos 182: 12.<br />
Barrios AM. 1992. Producci<strong>on</strong> y virulencia de algunas cepas del h<strong>on</strong>go<br />
en<strong>to</strong>mopa<strong>to</strong>geno <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Bals.) Vuill. c<strong>on</strong>tra la broca del<br />
cafe<strong>to</strong> Hypothenemus hampei (Ferrari). SIMPOSIO sobre Caficultura<br />
Latinoamericana 16: 25–29 Octubre 1993. Turrialba (Costa Rica),<br />
IICA-CATIE.<br />
Bateman RP.Lomer C, Lomer CJ. 1995. Formulati<strong>on</strong> and applicati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> mycopathogens for locust and grasshopper c<strong>on</strong>trol. LUBILOSA<br />
Technical Bulletin 4: 67.<br />
Bateman R. 2007. <str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g> Processes for Anamorphic Fungi<br />
IPARC, Silwood Park. Ascot, Berks. 16pp.<br />
Available <strong>on</strong>line at: http://www.dropdata.net/mycoharvester/<br />
Mass_producti<strong>on</strong>.PDF<br />
Bustillo AE, Posada FJ. 1996. El uso de en<strong>to</strong>mopatógenos en el c<strong>on</strong>trol<br />
de la broca del café en Colombia. Manejo Integrado de Plagas (Costa<br />
Rica) 42: 1-13.<br />
De La Rosa W, Ala<strong>to</strong>rre R, Barrera JF, Toriello C. 2000. Effect <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> and Metarhizium anisopliae (Deuteromycetes) up<strong>on</strong><br />
the c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer (Coleoptera:Scolytidae) under field c<strong>on</strong>diti<strong>on</strong>s.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Ec<strong>on</strong>omic En<strong>to</strong>mology 96(5): 1409-1414.<br />
Fernández PM, Lecu<strong>on</strong>a RE, Alves S. 1985. Pa<strong>to</strong>genicidade de <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g><br />
<str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Bols) Vuill a broca do cafe Hypothenemus hampei (Ferrari<br />
1867) (Coleoptera: Scolytidae). Ecossistema (Brasil) 10: 176-182.<br />
Ferr<strong>on</strong> P. 1978. Biological c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> insect pests by en<strong>to</strong>mogenous<br />
fungi. Annual Review <str<strong>on</strong>g>of</str<strong>on</strong>g> En<strong>to</strong>mology 23: 409-442.<br />
G<strong>on</strong>zález MT, Posada FJ, Bustillo AE. 1993. Desarrollo de un<br />
bioensayo para evaluar la pa<strong>to</strong>genicidad de <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> sobre<br />
Hypothenemus hampe. Revista Colombiana de En<strong>to</strong>mologia (Colombia)<br />
19(4): 123-130.<br />
Hajek AE. New opti<strong>on</strong>s for insect c<strong>on</strong>trol using fungi. In: Lumsden RD,<br />
Vaughn JL, edi<strong>to</strong>rs. 1993. Pest Management Biologically Based Technologies<br />
54-62. Washingt<strong>on</strong>, D.C., American Chemical Society.<br />
Haraprasad N, Niranjana SR, Prakash HS, Shetty HS, Wahab S. 2001.<br />
<str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> a potential mycopesticide for the efficient c<strong>on</strong>trol<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> c<str<strong>on</strong>g>of</str<strong>on</strong>g>fee berry borer, Hypothenemus hampei (Ferrari) in India. Bioc<strong>on</strong>trol<br />
Science and Technology 11(2): 251-260.<br />
Hussey WW, Tinsley TW. Impressi<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> insect pathology in the<br />
people’s Republic <str<strong>on</strong>g>of</str<strong>on</strong>g> China. In: Burges HD, edi<strong>to</strong>r. 1981. Microbial<br />
C<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Pest and Plant Diseases 1970–1980 785-795. Academic<br />
Press Inc.<br />
Jenkins NE, Goettel MS. Methods for mass- producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> microbial<br />
c<strong>on</strong>trol agents <str<strong>on</strong>g>of</str<strong>on</strong>g> grasshoppers and locusts. In: Goettel MS, Johns<strong>on</strong><br />
DL, edi<strong>to</strong>rs. 1997. Microbial C<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Grasshoppers and Locusts<br />
37-48. Memoirs <str<strong>on</strong>g>of</str<strong>on</strong>g> the En<strong>to</strong>mological Society <str<strong>on</strong>g>of</str<strong>on</strong>g> Canada #171.<br />
Jenkins NE. 1995. Studies <strong>on</strong> mass producti<strong>on</strong> and field efficacy <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
Metarhizium flavoviride for biological c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> locusts and grasshoppers.<br />
PhD thesis. Cranfield University, UK.<br />
J<strong>on</strong>es KA, Burges HD. Product stability: from experimental preparati<strong>on</strong><br />
<strong>to</strong> commercial reality. In: Evans HF. 1997. Microbial Insecticides:<br />
Novelty or Necessity? 163-171. BCPC Symposium proceedings<br />
#68.<br />
Lazo RR. 1990. Susceptibilidad de la broca del fru<strong>to</strong> del cafe<strong>to</strong> Hypothenemus<br />
hampei al h<strong>on</strong>go en<strong>to</strong>mopa<strong>to</strong>geno <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g>, y su<br />
<strong>to</strong>lerancia al oxicloruro de cobre. Turrialba (Costa Rica), IICA -<br />
CATIE (Tesis: Magister Scientiae).<br />
Lomer CJ, Prior C, Kooyman C. Development <str<strong>on</strong>g>of</str<strong>on</strong>g> Metarhizium sp. for<br />
the c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> grasshoppers and locusts. In: Goettel MS, Johns<strong>on</strong><br />
DL, edi<strong>to</strong>rs. 1997. Microbial C<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Grasshoppers and Locusts. 265-<br />
286. Memoirs <str<strong>on</strong>g>of</str<strong>on</strong>g> the En<strong>to</strong>mological Society <str<strong>on</strong>g>of</str<strong>on</strong>g> Canada #171.<br />
Marín P, Posada FJ, G<strong>on</strong>záles MT, Bustillo AE. 2000. Calidad biológica<br />
de formulaci<strong>on</strong>es de <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> usadas en el c<strong>on</strong>trol de<br />
la broca del café Hypothenemus hampei (Ferrari). Revista Colombiana de<br />
En<strong>to</strong>mología 26: 17-23.<br />
Mendez I. 1990. C<strong>on</strong>trol microbiano de la broca del fru<strong>to</strong> del cafe<strong>to</strong><br />
Hypothenemus hampei Ferrari (Coleoptera Scolytidae), c<strong>on</strong> el h<strong>on</strong>go<br />
<str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> (Bals.) Vuill. (Deuteromycetes) en el Soc<strong>on</strong>usco, Chiapas.<br />
Chapingo (Mexico), Universidad de Chapingo, Colegio de Postgraduados,<br />
(Tesis: Maestro en Ciencias).<br />
Moore D, Caudwell RW. Formulati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> en<strong>to</strong>mopathogens for the<br />
c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> grasshoppers and locusts. In: Goettel MS, Johns<strong>on</strong> DL,<br />
edi<strong>to</strong>rs. 1997. Microbial C<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Grasshoppers and Locusts 49-67.<br />
Memoirs <str<strong>on</strong>g>of</str<strong>on</strong>g> the En<strong>to</strong>mological Society <str<strong>on</strong>g>of</str<strong>on</strong>g> Canada #171.<br />
Morales E, Knauf W. C<strong>on</strong>idia ® , a formulati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> adapted<br />
for integrated pest management programs. VI Internati<strong>on</strong>al<br />
Colloquium <strong>on</strong> Invertebrate Pathology and Microbial C<strong>on</strong>trol. Abstracts pp.<br />
87. M<strong>on</strong>tpellier, France, 28 August- 2 September 1994. Volume<br />
II.<br />
Posada FJ, Villalba DA, Bustillo AE. 2004. Los insecticidas y el h<strong>on</strong>go<br />
<str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g> en el c<strong>on</strong>trol de la broca del café. Cenicafé (Colombia)<br />
55(2): 136-149.<br />
Posada FJ. C<strong>on</strong>trol biológico de la broca del café Hypothenemus hampei<br />
(Ferrri) c<strong>on</strong> h<strong>on</strong>gos. Memorias C<strong>on</strong>greso Sociedad Colombiana de En<strong>to</strong>mología,<br />
pp. 137–151. SOCOLEN, 20. Cali, Colombia, Julio 13–16<br />
1993.<br />
Posada FJ. 1998. <str<strong>on</strong>g>Producti<strong>on</strong></str<strong>on</strong>g>, formulati<strong>on</strong> and applicati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>Beauveria</str<strong>on</strong>g> <str<strong>on</strong>g>bassiana</str<strong>on</strong>g><br />
for c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Hypothenemus hampei in Colombia Ascot, Berkshire.<br />
University <str<strong>on</strong>g>of</str<strong>on</strong>g> L<strong>on</strong>d<strong>on</strong>. 227pp.<br />
Rao NK, Hans<strong>on</strong> J, Dulloo ME, Ghosh K, Nowell D, Larinde M.<br />
2006. Manual <str<strong>on</strong>g>of</str<strong>on</strong>g> seed handling in genebanks. Handbooks for Genebanks<br />
No. 8 Bioversity Internati<strong>on</strong>al. Rome, Italy163pp.<br />
Ruíz R. 1996. Efec<strong>to</strong> de la fenología del fru<strong>to</strong> del café sobre los parámetros<br />
de la tabla de vida de la broca del café, Hypothenemus hampei<br />
(Ferrari). Facultad de Ciencias Agropecuarias (Tesis Ing. Agrónomo)87<br />
Universidad de CaldasManizales, Colombia<br />
SAS Institute 2003. SAS/Stat user’s guide, versi<strong>on</strong> 8. SAS Institute Inc.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 12
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science | www.insectscience.org ISSN: 1536-2442<br />
Sp<strong>on</strong>agel KW. 1994. La broca del café Hypothenemus hampei en plantaci<strong>on</strong>es de<br />
café Robusta en la Amaz<strong>on</strong>ía Ecua<strong>to</strong>riana Giessen (Alemania). Wissenschaftlicher<br />
Fachverlag. 185pp.<br />
Thomas MB, Wood SN, Langewald J, Lomer CJ. 1997. Persistence <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
Metarhizium flavoviride and c<strong>on</strong>sequences for biological c<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
grasshoppers and locusts. Pesticide Science 49: 47-55.<br />
Vélez PE, Posada FJ, Marín P, Bustillo AE, G<strong>on</strong>zález MT, Osorio E.<br />
1997. Técnicas para el c<strong>on</strong>trol de calidad de formulaci<strong>on</strong>es de<br />
h<strong>on</strong>gos en<strong>to</strong>mopatógenos. Boletin Técnico 17: 37. Cenicafé,<br />
Colombia.<br />
Ye SD, Ying SH, Chen C, Feng MG. 2006. New solid-state fermentati<strong>on</strong><br />
chamber for bulk producti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> aerial c<strong>on</strong>idia <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>fungal</str<strong>on</strong>g><br />
bioc<strong>on</strong>trol agents <strong>on</strong> <strong>rice</strong>. Biotechnology Letters 28: 799-804.<br />
Yendol WG, Roberts DW. 1970. Is microbial c<strong>on</strong>trol with en<strong>to</strong>mogenous<br />
fungi possible? Society for Invertebrate Pathology. IVth Internatinal<br />
Colloquium with the Society for Invertebrate Pathology 28-42. College<br />
Park, Maryland USA. 25–28 August 1971.<br />
Journal <str<strong>on</strong>g>of</str<strong>on</strong>g> Insect Science: Vol. 8 | Article 41 13