DETERMINATION OF PATHOGENICITY AND VIRULENCE OF A COMBINATION OF STRAINS OF Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus OF THE COMPANY SANOPLANT, ON THE PSYLLID Diaphorina citri VECTOR OF THE HLB DISEASE OF CITRUS

1. 1
DETERMINATION OF PATHOGENICITY AND VIRULENCE OF A COMBINATION OF
STRAINS OF Beauveria bassiana, Metarhizium anisopliae and Paecilomyces
fumosoroseus OF THE COMPANY SANOPLANT, ON THE PSYLLID Diaphorina citri
VECTOR OF THE HLB DISEASE OF CITRUS
Carlos Aníbal Montoya
1
Eliana Andrea Rincón;
2
; Carlos Andrés Montoya
3
www.sanoplant.com.co
ABSTRACT
With the purpose of determining the pathogenicity and virulence of a combination of strains of Beauveria bassiana, Metarhizium
anisopliae and Paecilomyces fumosoroseus of the Company Sanoplant, on Diaphorina citri adults (vector of Candidatus
Liberibacter, the causative agent of HLB Greening or Huanglonbing), the laboratory evaluated the percentage of mortality, time of
infection and percentage of infection caused by the following treatments: 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), on D. citri adults arranged individually in Petri dishes, and a total of twenty (20) experimental units per treatment.
Seventy-two (72) hours after inoculation (ddi), Treatment 4 killed and mummified 100% of treated individuals. At 96 hours of ddi, the
mortality percentages of Treatments 2 and 3 were 100% and 10% for Treatment 1; 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 combination 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, a Hemiptero belonging to the Psyllidae family, is the vector of Candidatus Liberibacter
bacteria, the causative agent of HLB Greening or Huanglonbing, one of the most devastating diseases of
citrus fruit in the world (ICA, 2014).
Although Colombia has the susceptible host, the vector insect, it is unknown if the environmental factors or
the absence of the bacteria are the cause for not having the disease in the country.
Considering the disease triangle is almost closed and therefore can constitute a problem of economic
relevance (Agrios, 1997), it is necessary to have preventive methodologies for the control of the vector
insect, which are efficient, economic and environmentally feasible.
The use of fungi for the control of insect pests is a practice that has become more frequent in the country
(McCoy et al, 1992), thanks to the permanent effect they cause in insect pest populations, the
environmental safety, the global demand of green technologies, and the advanced research made at
national level by institutions such as Cenicafé, Ciat and Corpoica, as well as the manufacturers of biological
products that during almost fifty years have promoted biological control in Colombia and other countries.
The reproductive structures of the fungi, upon contact with the insect, adhere, germinate and penétrate
through physical and chemical mechanisms. Once inside, the fungus causes the insect’s death due to
nutritional deficiency, tissue destruction and toxins release (Bustillo, 2002). Other authors report that the
efficiency of pest control increased with a combination of strains of the same species, reaching a greater
effect when the combination is composed of low virulence strains (Cárdenas et al, 2007; Wang et al, 2004).
1
General Manager, Sanoplant. Agricultural bio-products manufacturing company. Calle 47 No. 30B32 Palmira Valle
2
Head of Laboratory, Sanoplant. Agricultural bio-products manufacturing company. Calle 47 No. 30B32 Palmira Valle
3
Production Manager. Sanoplant. Agricultural bio-products manufacturing company. Calle 47 No. 30B32 Palmira Valle

2. 2
Bustillo (2002), reports that in Colombia 42 species of pathogenic insect fungi have been found, with higher
occurrence of Beauveria bassiana and Metarhizium anisopliae. Likewise, the broad spectrum of
Paecilomyces fumoso-roseus is widely known.
The present research was done in order to evaluate the pathogenicity and virulence of a combination of
strains of Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus, active
ingredients of commercial products Beauveriplant, Metarhiplant and Paeciloplant, over the citrus Psyllid,
Diaphorina citri.
MATERIALS AND METHODS
D. citri adults from the artificial breeding in Tuluá, Valle, were inoculated in the laboratory of the agricultural
bio-products manufacturing Company, 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 active components of lots of commercial products denominated
Beauveriplant, Metarhiplant and Paeciloplant, respectively.
For the preparation of the inoculum, 5 ml of sterile distilled water were added to the Petri dishes with the
pure culture of the strain and with a sterile bacteriological broom, it was scraped in order to suspend the
greatest amount of growth of the microorganism in the distilled water. With the help of a micropipette, the
inoculum suspension was harvested and was served in a beaker, where it was increased to a volume of 10
ml, adding sterile distilled water. Test tubs with 9 ml of distilled water were used to make dilutions of 1 ml of
the inoculum suspension, until obtaining a dilution of 1x10-2
. Aliquots were taken into the Neubauer
chamber, to determine the concentration of conidias/ml., by calculating the average number of conidias
found in two (2) counts, multiplying it by the reverse of the count dilution (1x102
) and the ratio factor of the
chamber (1x104
) (Marín et al, 2002).
The inoculation of each treatment (Table 1) was done through the immersion of the insect in a suspension
of inoculum in sterile distilled water during one (1) minute, at a concentration of 1x107
conidias/ml.
Table 1. Treatments
Treatment Description
1 Negative Control Sterile distilled water
2 Beauveriplant SpBb59, SpBb60, SpBb25
3 Metarhiplant SpMa26, SpMa23
4 Paeciloplant SpPl3, SpPl6, SpPf18

3. 3
Figure 1. Inoculated insects
Insects were individually placed in the Petri dishes with sterile filter paper, were sealed and incubated at
± 25.6°C and 32% relative humidity. To discard death by starvation, myrtle hearts were placed in the dishes,
decontaminated with sterile distilled water (triple immersion). Each Petri dish was hydrated on a daily basis
with 100 µl of sterile distilled water. Twenty (20) experimental units were used in each treatment.
Daily mortality was registered, as well as the growth of fungi on the insects. This data was used to analyze
the response variables of the mortality percentage (PM), which corresponds to the ratio between the total
number of individuals and the number of individuals killed by the treatment action; infection time (TI), time
elapsed between the inoculation and the appearance of the reproductive structures of the inoculated
microorganisms; and the percentage of infection or the ratio between the total number of individuals and the
number of individuals with fungus growth.
Through a variance analysis (ANOVA), and Tukey’s Multiple Comparison Test of Means at a level of 5%,
the treatments with the higher percentage of mortality, lower time of infections and higher percentage of
infection, were selected.
RESULTS
- 72 hours after inoculation
At 72 hours after inoculation, significant statistical differences were found in the percentage of mortality, in
favor of Treatment 4, which resulted in a mortality percentage of 100%, followed by Treatments 3 and 2,
with 90% and 70%, respectively. The control treatment showed a mortality percentage of 10% (Table 2).
Table 2. Mortality percentage average 72 hours after inoculation (Tukey 5% - R
2
62%)
Treatment Description Mean N
1 Negative control 10 20 a
2 Beauveriplant 70 20 b
3 Metarhiplant 90 20 b
4 Paeciloplant 100 20 c
The different letters indicate significant differences (p<=0.05)
Seventy per cent (70%) of the insects under Treatment 4 were found mummified by the structures of the
inoculated microorganism 72 hours after inoculation. The percentage of infection of Treatments 3 and 2
was 55% and 40%, respectively. The statistical differences occurred between the mentioned treatments
and Treatment 1, which had an infection percentage of zero (Table 3).
Table 3. Adults infection percentage average with Diaphorina citri 72 hours after
inoculation (Tukey 5% - R
2
62%)
Treatment Description Mean N
1 Negative control 0 20 a
2 Beauveriplant 40 20 B
3 Metarhiplant 55 20 b
4 Paeciloplant 70 20 b
The different letters indicate significant differences (p<=0.05)
- 96 hours after inoculation
Ninety-six (96) hours after inoculation, 100% of the individuals of treatments 2, 3 and 4 were found dead;
this is statistically different from the percentage of mortality of treatment 1, which was 10% (Table 4)

4. 4
Table 4. Mortality percentage average 96 hours after inoculation (Tukey 5% - R
2
=
87%)
Treatment Description Mean N
1 Negative Control 10 20 a
2 Beauveriplant 100 20 b
3 Metarhiplant 100 20 b
4 Paeciloplant 100 20 b
The different letters indicate significant differences (p<=0.05)
There were no significant statistical differences in the infection percentage 96 hours after inoculation
between Treatments 4, 3 and 2, with infections percentages of 100%, 100% and 95%, respectively. The
infection percentage in Treatment 1 is zero (Table 5).
Table 5. Adults infection percentage average of Diaphorina citri 96 hours after
inoculation (Tukey 5% - R
2
87%)
Treatment Description Mean N
1 Negative Control 0 20 a
2 Beauveriplant 95 20 b
3 Metarhiplant 100 20 b
4 Paeciloplant 100 20 b
The different letters indicate significant differences (p<=0.05)
The following Figures show individuals under each Treatment, 96 hours after inoculation.
Figura 2. Individuals in Treatment 1 (negative control)

5. 5
Figure 3. Individuals in Treatment 2 (Beauveriplant)

6. 6
Figura 4. Individuals in Treatment 3 (Metarhiplant)

7. 7
Figura 5. Individuals in Treatment 4 (Paeciloplant)
CONCLUSIONS
Paecilomyces fumosoroseus in combination controlled and mummified 100% of treated individuals. The
percentages of mortality of the combination of Beauveria bassiana and Metarhizium anisopliae strains
were 100% and the infection percentages reached percentages over 95%.
The incubation period started after 48 hours. The mortality and latency period (sporulation over the
insects) started 96 hours after inoculation.
The combination of B. bassiana, M. anisopliae and P. fumosoroseus strains are an efficient alternative to
control Diaphorina citri, without affecting the biological equilibrium. It is important to highlight the

8. 8
contribution of this type of biological research in order to close the gap represented by this serious
phytosanitary problem at global level.
The results of this research provide an ecological and economic alternative to control D. citri before
environmental conditions enable the development of HLB or the development of Candidatus Liberibacter,
HLB causative agent, which is disseminated by this vector insect.
When using the combination of B. bassiana, M. anisopliae and de P. fumosoroseus in nurseries, the
dissemination of HLB is avoided and a low cost alternative, with low environmental impact is provided to
control the most devastating disease of citrus in the world.
Entomopathogenic fungi establishes in the agro-ecosystems in which they are sprayed and the insects
do not develop resistance characteristics to them, turning into an efficient alternative to control
Diaphorina citri.
Using biological control as a component of the integrated pest management (IPM) increases the
efficiency of the control of the crop's sanitary problems, without causing negative impacts on the agro-
ecosystem and generating lower production costs, improving profitability.
BIBLIOGRAPHY
AGIOS, G. N. 1997. Plant Pathology. 4th
edition. Academic Press. 636 p.
BUSTILLO, A. E. 2002. The use of fungal spores to control coffee berry borer. Hypothenemus hampei
(Ferrari). In memoirs of a practical-theoretical international course on entomopathogens, parasites and other
enemies of coffee berry borer. Chinchiná, Caldas. March 11-15, 2002. p 54-64.
CARDENAS, R.; VILLALBA, D. A.; BUSTILLO, A. E.; MONTOYA, E.C.; GONGORA, C. E. 2007. Efficiency
of the combination of strains of fungus Beauveria bassiana in the control of coffee berry borer. Cenicafe 58
(4): 293-303.
INSTITUTO COLOMBIANO AGROPECUARIO ICA. 2014. Exotic diseases and pests of the citrus sector in
Colombia D. C. Kuwayama. http://www.ica.gov.co/getdoc/77e880b7-40d6-4144-a398-
e7dd3a855237/DIAPHORINA.aspx
MARIN, P.; BUSTILLO, A. E. 2002. Microbiological and physical-chemical tests for quality control of
entomapathogenic fungi. In memoirs of a practical-theoretical international course on entomopathogens,
parasites and other enemies of coffee berry borer. Chinchiná, Caldas. March 11-15, 2002. p 72-89.
McCOY, C. W.; TIGANO, M. S. 1992. Use of entomopathogenic fungi in biological control: a world view.
Pesquisa 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 Microbiology 96:
861-870.