This document evaluates the pathogenicity and virulence of mixtures of Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces fumosoroseus fungal strains from the company Sanoplant for controlling the psyllid Diaphorina citri, which vectors the bacterium causing Huanglongbing (HLB) disease in citrus. Adults were inoculated with the fungal formulations Beauveriplant, Metarhiplant, and Paeciloplant. Paeciloplant achieved 100% mortality within 72 hours, while Beauveriplant and Metarhiplant reached 100% mortality and over 95% infection rates within

1. DETERMINATION of the PATHOGENICITY and VIRULENCE of mixture of strains of
Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus of the
company SANOPLANT, on the PSYLLID Diaphorina citri
VECTOR OF THE DISEASE HLB OF THE CITRUS
Carlos Aníbal Montoya1
Eliana Andrea Rincón;2
; Carlos Andrés Montoya3
www.sanoplant.com.co
RESUMEN
Con el objetivo de determinar la patogenicidad y virulencia de mezcla de cepas de Beauveria bassiana, Metarhizium anisopliae y
Paecilomyces fumosoroseus de la Empresa Sanoplant, sobre adultos de Diaphorina citri (vector de Candidatus Liberibacter,
agente causante del HLB, Enverdecimiento o Huanglonbing), en laboratorio se evaluaron el porcentaje de mortalidad, tiempo de
infección y porcentaje de infección, causada por los tratamientos: inoculación con Beauveriplant (tratamiento 2), inoculación con
Metarhiplant (tratamiento 3) e inoculación con Paeciloplant (tratamiento 4), frente a un testigo absoluto (tratamiento 1, agua
destilada estéril), sobre adultos de D. citri dispuestos de manera individual en Cajas de Petri, y un total de 20 unidades
experimentales por tratamiento. A las 72 horas después de la inoculación (ddi), el tratamiento 4 mató y momificó el 100% de los
individuos tratados. A las 96 horas ddi, los porcentajes de mortalidad de los tratamientos 2 y 3 fueron del 100% y del tratamiento 1
fue del 10%; y el porcentaje de infección fue superior al 95% en los tratamientos 2 y 3, y de cero en el tratamiento 1. Bajo las
condiciones de este estudio se concluye que la mezcla de cepas seleccionadas de las especies Beauveria bassiana,
Metarhizium anisopliae y Paecilomyces fumosoroseus son rápidas y eficaces en el control de D. citri.
ABSTRACT
With the objective of determining the pathogenicity and virulence of mixture of strains of Beauveria bassiana, Metarhizium
anisopliae and Paecilomyces fumosoroseus the company s Sanoplant on adults ofʹ Diaphorina citri (Candidatus Liberibacter
vector of agent which causes the HLB Greening or Huanglonbing), the percentage of mortality, time of infection and percentage of
infection, caused by the treatments were evaluated in laboratory: inoculation with Beauveriplant (treatment 2) inoculation with
Metarhiplant (treatment 3) and inoculation with Paeciloplant (treatment 4), against an absolute control (treatment 1 sterile distilled
water), adults of D. citri arranged individually in Petri dishes, and a total of 20 experimental units for treatment. 72 hours after
inoculation (ddi), treatment 4 killed and mummified 100% of treated individuals. 96 hours ddi treatments 2 and 3 mortality
percentages were 100% and treatment 1 was 10%; and the percentage of infection was higher than 95% in treatments 2 and 3; and
zero in treatment 1. Under the conditions of this study, it is concluded that the mixture of selected strains of the species, Beauveria
bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus are quick and efficient in the control of D. citri.
______________________________________________________________________________________
Diaphorina citri, bug of the family Psyllidae, is the vector of the bacterium Candidatus Liberibacter, agent
causing the HLB, greening or Huanglonbing, one of the most devastating diseases of citrus in the world
(ICA, 2014).
Colombia has with the susceptible host, the insect vector, but it is unknown whether they are environmental
factors or the absence of bacteria, causing that disease is not present in the country.
Taking into account that disease triangle is almost already closed, and thus it may constitute a problem of
economic importance (Agrios, 1997), it is necessary to have preemptively with methodologies for the control
of vector insect that are effective, economic and environmentally viable.
The use of fungi for control of insects plague is a practice that has caught more and more strength in the
country, (McCoy et al, 1992), thanks to the permanent effects that cause plague, safety in populations of
insects with the environment, global demands for clean technologies and advances in research to national
level institutions such as Cenicafé, Ciat Corpoica and producers of biological supplies that for nearly 50
years have touted the Biological Control in Colombia and other countries.
1
Gerente General Sanoplant. Empresa Productora de Bioinsumos Agrícolas. Calle 47 No. 30B32 Palmira Valle
2
Jefe de laboratorio Sanoplant. Empresa Productora de Bioinsumos Agrícolas. Calle 47 No. 30B32 Palmira Valle
3
Jefe de producción. Sanoplant. Empresa Productora de Bioinsumos Agrícolas. Calle 47 No. 30B32 Palmira Valle
1

2. The structures reproductive of the fungus, to the enter in contact with the insect, is stick, germinate and
penetrate through mechanisms physical and chemical. Within the insect fungus causes his death by
nutritional deficiency, destroying tissue and release toxins (Bustillo, 2002). Other authors reported that pest
control efficiency increases when applying mixture of strains of the same species, the effect being greater
when the mixture to make it up strains of low virulence (Cárdenas et al, 2007; Wang et al, 2004).
Bustillo (2002), reporta que en Colombia se han encontrado 42 especies de hongos patógenos de insectos,
siendo las de mayor ocurrencia Beauveria bassiana y Metarhizium anisopliae. De igual manera, no se
desconoce el amplio espectro del hongo Paecilomyces fumoso-roseus.
Bustillo (2002), reports that in Colombia is have found 42 species of fungal pathogens of insects, being
them of greater occurrence Beauveria bassiana, Metarhizium anisopliae y Paecilomyces
fumosoroseus, ingredients active of those products commercial Beauveriplant, Metarhiplant and
Paeciloplant, on the Psyllid of them citrus Diaphorina citri.
MATERIALS AND METHODS
Adults of D. citri of the breeding artificial in Tulua Valley, were inoculated in the laboratory of the company
producer of bioinputs agricultural Sanoplant, with Beauveria bassiana (strains SpBb59, SpBb60 and
SpBb25), Metarhizium anisopliae (strains SpMa26 and SpMa23) and Paecilomyces fumoso-roseus (strains
SpPl3, SpPl6 and SpPf18), as components active of lots of the products commercial Beauveriplant,
Metarhiplant and Paeciloplant respectively.
To prepare the inoculum, 5 ml of sterile distilled water sterile Petri boxes with the pure culture of the strain,
and bacteriological rake is added to, a scraping became so much of microorganism growth is suspended in
distilled water. With the help of a micropipette, inoculum suspension was harvested and served in a beaker,
where took a volume of 10 ml, adding sterile distilled water. In tubes of essay with 9 ml of water distilled, is
made dilutions of 1 ml of the suspension of inoculum, until get a dilution of 1 by 10-2, of which is carried
aliquots to the camera of Neubauer, to determine the concentration of conidia / ml. Which, we calculated the
average number of conidia were found on two counts, was multiplied by the inverse of the dilution of count
(1 x 102) and camera correlation factor (1 x 104) (Marín et al, 2002).
The inoculation of each treatment (table 1), is made by the immersion of them insects during 1 minute in a
suspension of inoculum in water distilled sterile to a concentration of 1 by 107 conidia / ml.
Tabla 1. Tratamientos
2

3. Figure 1. Inoculated insects
Insects were individually placed in Petri dishes with sterile filter paper, sealed and drove to incubation at
25.6 ° c and 32% of relative humidity. To rule out a death by starvation, buds of Myrtle disinfested with
sterile distilled water (triple immersion) were placed inside boxes. Daily every box of Petri is hydrated with
100 µl of water distilled sterile. For each treatment were 20 experimental units.
Is recorded the mortality daily, similar to the growth of the fungus on the insects. With these data is analyzed
the variables of response percentage of mortality (PM), that corresponds to the relationship between the
number total of individuals and the number of individuals dead by the action of the treatment; time of
infection (TI), elapsed time between inoculation and the onset of the reproductive structures of
microorganisms inoculated; and the percentage of infection or relationship between the total number of
individuals and the number of individuals with growth of the fungus.
Using an analysis of variance (Anova), and Multiple comparison of Tukey mean test to the 5% level, higher
percentage of mortality, less time of infection and highest percentage of infection treatments were selected.
RESULTS
- 72 hours after inoculation
72 hours after inoculation were found no statistical difference in the percentage of mortality in favour of
treatment 4 presented a percentage of 100% mortality, followed by treatments 3 and 2, with rates of 90%
and 70%, respectively. Treatment control featured a 10% mortality (table 2).
Table 2. Average of the percentage of mortality at 72 hours after inoculation (Tukey
5% - R2
62%)
Tratamiento Descripción Media N
1 Control negativo 10 20 a
2 Beauveriplant 70 20 b
3 Metarhiplant 90 20 b
4 Paeciloplant 100 20 c
Different letters indicate significant differences (p < = 0,05)
70% of treatment 4 insects were found mummified by the structures of the organism inoculated within 72
hours after inoculation, the infection of treatments 3 and 2 percentage was 55% and 40%, respectively.
Statistical differences arose between the above treatments and 1, whose infection rate was zero (table 3).
Table 3. Average of the percentage of adult infection of Diaphorina citri 72 hours
after inoculation (Tukey 5% - R2
62%)
3

4. Tratamiento Descripción Media N
1 Control negativo 0 20 a
2 Beauveriplant 40 20 B
3 Metarhiplant 55 20 b
4 Paeciloplant 70 20 b
Different letters indicate significant differences (p < = 0,05)
70% of treatment 4 insects were found mummified by the structures of the organism inoculated within 72
hours after inoculation, the infection of treatments 3 and 2 percentage was 55% and 40%, respectively.
Statistical differences arose between the above treatments and 1, whose infection rate was zero (table 3).
Table 3. Average of the percentage of adult infection of Diaphorina citri 72 hours
after inoculation (Tukey 5% - R2
62%)
Tratamiento Descripción Media N
1 Control negativo 10 20 a
2 Beauveriplant 100 20 b
3 Metarhiplant 100 20 b
4 Paeciloplant 100 20 b
Different letters indicate significant differences (p < = 0,05)
Significant statistical differences in the rate of infection was no 96 hours after inoculation treatments 4, 3 and
2, with rates of infection of 100%, 100% and 95%, respectively. The percentage of infection in treatment 1 is
zero (table 5).
Table 5. Average of the percentage of infection of adults of Diaphorina citri to the
96 hours after the inoculation (Tukey 5%-R2
87%)
Tratamiento Descripción Media N
1 Control negativo 0 20 a
2 Beauveriplant 95 20 b
3 Metarhiplant 100 20 b
4 Paeciloplant 100 20 b
Lyrics different indicate differences significant (p < = 0,05)
The following figures illustrate individuals of each treatment 96 hours after inoculation.
Figura 2. Individuos del tratamiento 1 (control negativo)
4

5. Figura 3. Individuos del tratamiento 2 (Beauveriplant)
Figura 4. Individuos del tratamiento 3 (Metarhiplant)
5

6. Figura 5. Individuos del tratamiento 4 (Paeciloplant)
CONCLUSIONES
Bajo las condiciones de este estudio se concluye que:
• Beauveriplant, Metarhiplant y Paeciloplant productos comerciales de la Empresa productora de
Bioinsumos Agrícolas SANOPLANT, son eficaces en el control de adultos de Diaphorina citri.
• A las 72 horas después de la inoculación, Paeciloplant mató el 100% de los individuos tratados,
momificándolos de igual manera en un 100%, lo que lo hace el tratamiento más virulento contra este
insecto plaga.
• Los porcentajes de mortalidad de Beauveriplant y Metarhiplant fueron del 100% y los porcentajes de
infección alcanzaron valores por encima del 95%, a las 96 horas después de la inoculación, lo que de
igual manera los hace patogénicos y virulentos sobre los adultos de Diaphorina citri.
BIBLIOGRAFIA
AGIOS, G. N. 1997. Plant Pathology. 4ta edición. Academic Press. 636 p.
BUSTILLO, A. E. 2002. El uso de hongos para el control de la broca del café. Hypothenemus hampei
(Ferrari). En memorias curso internacional teórico practico sobre entomopatógenos, parasitoides y otros
enemigos de la broca del café. Chinchiná Caldas. Marzo 11-15 de 2002. p 54-64.
6

7. CARDENAS, R.; VILLALBA, D. A.; BUSTILLO, A. E.; MONTOYA, E.C.; GONGORA, C. E. 2007. Eficacia de
mezcla de cepas del hongo Beauveria bassiana en el control de la broca del café. Cenicafe 58 (4): 293-303.
INSTITUTO COLOMBIANO AGROPECUARIO ICA. 2014. Plagas exóticas a la citricultura en Colombia D.
C. Kuwayama. http://www.ica.gov.co/getdoc/77e880b7-40d6-4144-a398-e7dd3a855237/DIAPHORINA.aspx
MARIN, P.; BUSTILLO, A. E. 2002. Pruebas microbiológicas y físico químicas para el control de calidad de
hongos entomopatógenos. En memorias curso internacional teórico practico sobre entomopatógenos,
parasitoides y otros enemigos de la broca del café. Chinchiná Caldas. Marzo 11-15 de 2002. p 72-89.
McCOY, C. W.; TIGANO, M. S. 1992. Use of entomopathogenic fungi in biological control: a wold view.
Pesquiza Agropecuaria Basilera 27: 87-93.
WANGH, C.S.; FAN, M. L.; BOTT, T. M. 2004. Molecular monitoring and evaluation of the application of the
insect – pathogenic fungus Beauveria bassiana in south east china. Journal of applied microbiologic 96:
861-870.
7