Sjavs 108 99-113

Scholars Journal of Agriculture and Veterinary Sciences
Abbreviated Key Title: Sch J Agric Vet Sci

ISSN 2348–8883 (Print) | ISSN 2348–1854 (Online)
Journal homepage: https://saspublishers.com
In Vitro Evaluation of Native Isolates of Lecanicillium spp (Berk &

Broome) on Hemileia vastatrix
1
Santos David Romero , Trinidad Castillo-Arévalo* 1
, Markelyn José Rodríguez Zamora 1
1
National Agrarian University, Department of Agricultural and Forestry Protection, Faculty of Agronomy, Km 12.5, Northern
Highway, Managua, Nicaragua
DOI: 10.36347/sjavs.2023.v10i08.001 | Received: 09.07.2023 | Accepted: 02.08.2023 | Published: 11.08.2023

*Corresponding author: Trinidad Castillo-Arévalo
National Agrarian University, Department of Agricultural and Forestry Protection, Faculty of Agronomy, Km 12.5, Northern
Highway, Managua, Nicaragua
Abstract Original Research Article
With the objective of developing biological management options for coffee rust, through the characterization and
evaluation of native isolates of Lecanicillium spp. The inoculum was obtained from hyperparasitized rust pustules and
purified in potato dextrose agar culture medium. Lecanicillium spp. isolates were studied by microscopic,
macroscopic, and physiological characterization, as well as In Vitro tests to determine the parasitism of each isolate on
uredospores and rust pustules, and the potential for mass production of conidia on organic substrates: soybean,
sorghum and rice. Six native isolates of the genus Lecanicillium spp. were obtained from 20 samples, with conidia
ranging in size from 2.5 to 7.5 µ in length and from 1.5 to 2 µ in width, in groups or solitary, hyaline in color and
cylindrical or ellipsoidal in shape. The size of the phialides ranged from 20 to 28 µ with a base width of 2.5 µ.
Phialides were found solitary or in whorls originating from straight conidiophores, the conidia generally in groups of
two to six, sometimes solitary. The average radial growth of Lecanicillium spp. 20 days after inoculation was 18 mm
in PDA, 15 mm in EMA and 16 mm in SDA. The Majada isolate presented the highest percentage of viability with
99.7% at 22 hours. The isolate La Gotera on the soybean-based organic substrate showed the highest conidial yield of
1.08E+09 per gram of substrate compared to the other substrates and the other isolates. The Jinotega isolate presented
the highest parasitism of uredospores in Petri dishes 16.6%. The highest percentage of parasitism on pustules was
presented by the isolate La Gotera with 88.3%.
Keywords: Biological control, hyperparasitized, pustules, parasitism, uredospores, uredospores.
Copyright © 2023 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (CC BY-NC 4.0) which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original
author and source are credited.
INTRODUCTION 2004). Pest control in coffee growing has been using

Coffee (Coffea arabica L.) is one of the most chemicals, but due to undesirable effects on beneficial
important crops in the world from an economic and organisms, groundwater, food safety and development
social point of view. For the Nicaraguan economy, of insect resistance (Shahid et al., 2012), these
coffee has been over the years the item that has had the strategies have not been the ideal solution to the
greatest contribution to the economy, representing problem (Javed, 1987). Many importing countries are
about 30% of agricultural GDP and 50% of foreign increasingly demanding specialty coffees such as
exchange from exports (Moraga et al., 2012). During organic coffee (Caixeta and Pedini, 2002). The price of
the 2011-2012 cycle Nicaragua had a coffee export of organic coffee can reach prices between four and five
450,383,446.61 dollars and for the 2012-2013 cycle it times higher than conventionally cultivated coffee. The
had an export of 430, 721,041.16 (CETREX, 2020), this demand for organic coffee requires certain
shows a decrease of 19,662,405.5 million dollars due to requirements, that it be free of certain pesticides that are
the high incidence of rust of 37.0%. The rust crisis has normally applied to control pests and diseases. It is
put the survival of hundreds of thousands of families in therefore necessary to develop organic production
the region at risk (Avelino et al., 2013). systems that benefit the environment and human health.
Rust (Hemileia vastatrix Berk. & Br.) is the Entomopathogenic fungi are distributed
most important disease of coffee whose damage can approximately in more than 100 genera and 750
cause defoliation between 30% and 50% (Avelino et al., species, some of which present agronomic interest as
microbial control agents (García et al., 2019). These
Citation: Santos David Romero, Trinidad Castillo-Arévalo, Markelyn José Rodríguez Zamora. In Vitro Evaluation of
Native Isolates of Lecanicillium spp (Berk & Broome) on Hemileia vastatrix. Sch J Agric Vet Sci, 2023 Aug 10(8): 99-113. 99
Santos David Romero et al., Sch J Agric Vet Sci, Aug, 2023; 10(8): 99-113
fungi include Lecanicillium spp, Beauveria bassiana, control of coffee rust (Leguizamón et al., 1989). Given
Metarhizium spp, Isaria fumosorosea and among others, the importance of coffee rust disease, the purpose of the
which are used for the control of dozens of pests in a present study is to contribute to the development of new
wide variety of crops (Castillo-Arévalo, 2022, Castillo alternatives for biological management of the disease
Arévalo, 2023). through characterization and evaluation under
laboratory conditions of native isolates of Lecanicillium
The use of biopesticides is a tool to improve spp on coffee rust.
strategies for the rational use of insecticides, including
microbial pesticides based on bacteria, fungi, viruses, MATERIAL AND METHODS
and nematodes (Bautista et al., 2010). The fungus The research was conducted at the biopesticide
Lecanicillium spp. is one of the most common laboratory of the National Agrarian University,
hyperparasites of coffee rust, occurs naturally in coffee Nicaragua in January 2018.
plantations and could be a good candidate for biological
The study was carried out in two stages, the was being isolated. Once confirmed by taxonomic keys
first consisted of collecting samples of Lecanicillium (Zare and Gams, 2001) and purified on PDA culture
spp. from leaves with rust (H. vastatrix) parasitized by medium.
Lecanicillium spp. for subsequent isolation and
characterization. In stage two, six native isolates of Macroscopic and physiological characterization of
Lecanicillium spp. were evaluated in laboratory Lecanicillium spp. isolates
conditions to determine the level of parasitism on This was done in three culture media, Potato
uredospores and on rust pustules on leaves from the Dextrose Agar (PDA), Sabouraud Dextrose Agar
field. The radial growth of all isolates was also (SDA) and Malt Extract Agar (EMA). This study also
evaluated in Petri dishes of 90 mm in diameter in potato included characteristics related to their mass production
dextrose agar, sabouraud dextrose agar and malt extract on natural substrates of rice, soybean, and sorghum.
agar, and they were also evaluated on different organic The germination reading of Lecanicillium spp. conidia
substrates to determine promising strains for mass was done at 24 hours and at least 200 conidia per Petri
reproduction. dish were quantified with a 40x objective. To proceed
with the characterization, the fungus was transferred
The methodology to isolate Lecanicillium spp. from a pure culture to the three-culture media in 90 mm
was dry isolation, which consisted of taking the diameter Petri dishes, in which the corresponding
inoculum directly from the leaves with parasitized rust observations were made and six native isolates of
pustules using a hypodermic needle and deposited in Lecanicillium spp were evaluated, using 10 replicates
Petri dishes containing acidified potato dextrose agar for each isolate in the three culture media for a total of
potato culture medium. The samples were previously 30 dishes per isolate and one Petri dish was considered
observed under the microscope, using staining with as a replicate.
lactophenol blue to determine that the desired fungus
© 2023 Scholars Journal of Agriculture and Veterinary Sciences | Published by SAS Publishers, India 100
Microscopic characterization Viability of conidia

The size of the reproductive structures of the It was determined by evaluating the average
fungus was measured, such as: size of the phialides, conidia germination time described by Monzon (2001).
size of the conidia and width of the base of the The methodology consisted of placing one gram of
phialides, as well as the shape and color of the conidia. fungus on rice substrate in a test tube containing 9 ml of
A previously calibrated LW-Scientific light microscope sterile distilled water, thus preparing serial dilutions 10-
was used for this measurement, using a 40x lens. The 1, 10-2, 10-3. Once the first dilution was prepared, it
data were taken 48 hours after inoculation of the culture was homogenized by vortexing at 3 thousand rpm and
medium and the number of structures taken was thirty kept for three minutes to separate the conidia from the
for each variable for each culture medium (PDA, SDA, substrate. From the first dilution, the following dilutions
and EMA). To avoid contamination with bacteria, a 2% were prepared, and the most appropriate dilution was
chloramphenicol-based antibiotic was used. selected to take the reading of the viability study. For
Macroscopic characteristics included qualitative aspects the establishment of the test, a 5 µl aliquot of the
such as colony color, growth type and growth rate. Lecanicillium spp suspension was deposited with a
micropipette in a 90 mm diameter Petri dish containing
Radial growth 1.5% Agar-water medium, autoclaved at 1.2 bar
This was done in 90 mm diameter Petri dishes pressure and 121°C temperature. The inoculated Petri
based on the methodology described by French and dishes were placed in a room at a temperature of 24°C
Hebert (1982). The technique consisted of placing the with eight hours of light per day.
inoculum in the center of the dish containing the culture
medium and then drawing a cross on the back of the Germination readings were taken at 16, 18 and
Petri dish; the center of the dish was the inoculation 22 hours, using a 40X LW-Scientific light microscope.
point; the cross delimits four radii identified with letters For this study, three Petri dishes were used for each
A, B, C and D on which the readings were taken in reading time, for a total of nine dishes for each isolate.
millimeters. The data were taken by measuring the At the time of viability reading in each dish 10
growth of the fungus on each of the four marked radii. microscopic fields were observed, these were
Measurements were taken every 48 hours for a period previously delimited with a marker on the back of the
of 20 days. Thirty plates were used for each isolation, dish; a total of thirty fields were observed for each
10 for each culture medium for a total of 180 isolate in each reading, observing at least 200 conidia in
experimental units. each dish. The viability percentage was calculated using
the following formula (French and Hebert, 1982);
germinated conidia was one whose germinative tube
reached twice its length.
( ) Equation 1
Mass Production manually homogenized to ensure uniform growth of the

Each isolate of Lecanicillium spp. was fungus; the bags were then placed in an incubation
evaluated by the production of conidia on rice, soybean, room at a temperature of 24°C in dark conditions.
and sorghum, based on a semi-industrial biphasic mass
production method. The methodology consisted of Performance evaluation was performed 20
reproducing the inoculum in a synthetic solid culture days after inoculation when the substrate had been fully
medium and in liquid matrices based on nitrogen, colonized and sporulated by the fungus. Of the 10 bags
vitamins, phytohormones, sucrose and skim milk. The of each isolate with a given organic substrate, five were
inoculum for inoculation of the natural solid substrates randomly selected for counting. For the readings, one
to be evaluated was taken from the matrix. Two gram was weighed in rice and serial dilutions (10-2, 10-
hundred grams of each raw substrate were placed in 3, 10-4, 10-5) were prepared and the 10-3 dilution was
thermos resistant bags and 50 ml of drinking water was selected for conidia counting. To homogenize and
added, then the bags were sealed with staples, then the separate the conidia from the substrate, the tubes were
bags were sterilized by moist heat at 1.5 bar pressure at shaken for three minutes in a vortex at 2400 rpm.
a temperature of 121oC for 5 minutes. Once the bags Conidia counting was performed in a Neubauer
with the substrates were sterilized and cooled, 10 bags chamber and an LW-Scientific microscope with a 40x
were inoculated with each isolate, depositing 20 ml of lens was used. For the calculation of yield number of
fungal suspension (Monzon, 2001). The inoculation of spores/gram of substrate, the following formula was
the bags was carried out in a laminar flow chamber. used:
After inoculation, the substrate inside the bags was
Equation 2
Parasitism In Vitro of Lecanicillium spp. on H. dishes were used for each isolate of Lecanicillium spp.
vastatrix 5µl of the uredospore suspension were deposited on the
For the study of parasitism of Lecanicillium surface of the medium in five points previously
spp on uredospores of H. vastatrix consisted of scraping delimited with a marker. Subsequently, 5 µl of the
with a scalpel on the underside of leaves in rust lesions suspension of conidia of Lecanicillium spp. was
free of the hyperparasite, then deposited in a container deposited on the spore suspension of H. vastatrix. Once
with water, and a concentration of 1x10-4 the test was established, the plates were placed in a
uredospores/ml was prepared in sterile distilled water. room at a temperature of 24°C. To determine the
The inoculum of Lecanicillium spp isolates was percentage of parasitism of Lecanicillium spp. on the
obtained from the growth of the fungus on rice uredospores, five spots per Petri dish were observed and
substrate, from that substrate a suspension of 1x10-5 a minimum of 50 uredospores were quantified. For the
spores/ml was prepared. The parasitism test was reading a LW-Scientific light microscope was used to
performed in Petri dishes of 90 mm in diameter determine parasitism using the following formula:
containing 1.5% Agar-water. For this study, three
…………… Equation 3
Parasitism of Lecanicillium spp on rust pustules chamber consisting of a plastic container 30 cm long x
A bioassay was established on coffee leaves 18 cm wide x 10 cm deep, with a 2.5 cm high mesh, on
under laboratory conditions to determine the which ten leaves were placed in each container and
effectiveness of Leacnicillium spp on rust pustules. each leaf was considered as a repetition. To maintain
Leaves with fresh rust pustules, highly sporulated and relative humidity greater than 95%, 500 ml of water
free of Lecanicillium spp. were collected. Before setting was placed at the bottom of each container. Finally, the
up the study, the samples were analyzed with mounts containers were placed in a dark chamber, formed with
and observed under the microscope to confirm the plastic and moistened craft paper in such a way that all
absence of the hyperparasite. To avoid the premature the leaves were covered with black plastic to ensure
aging process of the leaves and to keep them turgid for high humidity. The bioassay temperature ranged
120 hours during the evaluation period, a cotton swab between 22°C and 24°C and a relative humidity above
impregnated with 3 milliliters of a 2% solution of 6 95% inside the containers. For the evaluation of
BAP (Benzyl amino purine) at pH 5.8 was placed on parasitism of Lecanicillium spp. on the pustules,
the petiole of each leaf (Tórrez and Castillo, 2005). observations were made at 36 hours, the time required
for the hyperparasite to colonize the uredospores. For
Inoculation of Leacnicillium spp. on rust the calculation of the percentage of parasitism, mounts
pustules was done by immersion at a concentration of were made to observe mycoparasitism at the
1x10-8 conidia/ml. After immersing the leaves with rust microscopic level and the following formula was used
in the conidia suspension, it was left to stand for two (Monzon, 1992).
minutes, then the leaves were placed in a humid
( ) ………Equation 4
RESULTS Microscopic characteristics

A total of 20 samples were collected, from All Lecanicillium spp. isolates were similar in
which six isolates of Lecanicillium spp. were obtained, the three media evaluated. Conidia size was similar and
which were subjected to all the tests performed in the ranged from 2.5 to 7.5 µ in length and 1.5 to 2 µ in
study. All isolates were obtained from coffee plants; width, with conidia present at the end of the phialides in
these genetic materials are considered native isolates clusters or solitary, hyaline in color and cylindrical or
because no commercial applications of Lecanicillium ellipsoidal in shape (Figure 1). The size of the phialides
spp. had been made. ranged from 20 to 28 µ with a base width of 2.5 µ and
thinning at the end in a kind of very small tip, solitary
phialides were found or in whorls originating from
straight conidiophores, the conidia generally in groups
of two to six and sometimes solitary (Figure 1).
Figure 1: Reproductive structures of Lecanicillium spp. cylindrical, ellipsoidal, and rounded conidia (a).
Verticillate conidiophores with phialides and solitary conidia (b). Verticillate conidiophore with phialides and
conidia in clusters (c). Solitary phialides with conidia in clusters (d)
Macroscopic characteristics greater vertical growth in the upper part due to the
The growth of the colonies was similar in color abundant mycelium, in the lower part of the plate a
and shape, they presented an off-white coloration, with wrinkling or ridges were observed in the medium
a cottony appearance, in addition the colonies presented during their growth (Figure 2).
Figure 2: Lecanicillium spp. colonies in different culture media. PDA culture medium. Front view (A), back view
(B). SDA culture medium front view (C), rear view (D), EMA culture medium front view (D) rear view (F)
The average radial growth of all native isolates where the highest growth values were recorded,
of Lecanicillum spp achieved varied growth on the followed by the SDA culture medium, the lowest
culture media evaluated. According to Hall (1984) the growth values were recorded in the EMA culture
fungus L. lecanii grows well on almost any culture medium (Figure 3).
medium. In our study the PDA culture medium was
Figure 3: Average radial growth of Lecanicillium spp. isolates 20 days after inoculation on PDA, EMA, and SDA medium
The analysis of variance for the variable of by other isolates. Since the results were significant for
radial growth in EMA culture medium, indicates the interaction between the isolates and the evaluation
significant statistical differences between the evaluation periods for the EMA and PDA culture media, an
periods (p< 0.0001); it also indicates that there are analysis of variance was performed to compare the
significant differences (p< 0.0001) between isolates. radial growth of the isolates in each evaluation period.
Furthermore, it indicates that the Time*Isolate
interaction is significant (p= 0.002). This same result In the malt extract agar culture medium, the
was found in PDA culture medium. In the SDA culture San Ramón isolate showed the highest growth rate in
medium, the radial growth of Lecanicillium spp isolates most of the evaluation periods, while the Mankotal
was significant for the evaluation period factor isolate showed the lowest radial growth in all evaluation
(p<0.0001) and the isolate factor (p<0.0001). periods, except at 48 and 192 hours after inoculation.
The analysis of variance performed for the different
The results of the interaction between the two dates indicated differences in radial growth between
factors under study, isolates and evaluation periods for isolates at 48 (p<0.0001), 96 (p<0.0259), 192
the radial growth of Lecanicillium spp, in EMA and (p<0.0001), 240 (p<0.0039) and 336 hours (p<0.0002).
PDA, indicate that the behavior of the radial growth of Radial growth ranged from 0.31 mm to 3.16 mm. The
the isolates varies significantly through time in each San Ramón isolate recorded the highest growth with 3.6
isolate, that in some periods some isolates result with mm, while the Mankotal isolate recorded the lowest
greater radial growth, but in others they are surpassed radial growth with 0.31 mm.
Figure 4: Radial growth (mm) of Lecanicillium spp isolates at different times on malt extract agar culture medium
The analysis of variance for the radial growth from 0.71 mm to 2.98 mm every 48 hours. The isolate
of Lecanicillium spp isolates in PDA culture medium Majada presented the highest radial growth at 336
indicates that there are significant differences at 48 hours, while the isolate San Ramón presented the
(p<0.0001), 192 (p<0.0001) and 384 hours after lowest radial growth at 288 hours and in most of the
inoculation (p<0.0083). In this culture medium, the sampling periods. The isolate Mankotal presented the
radial growth values of the different isolates ranged best growth in most of the sampling periods (Figure 5).
Figure 5: Radial growth (mm) of Lecanicillium spp isolates at different times in potato dextrose agar medium
The radial growth of Lecanicillium spp in SDA hours. The Mankotal isolate presented the highest
was significant for the evaluation period factor average radial growth at 336 hours, while the San
(p<0.0001) and the isolate factor (p<0.0001), however, Ramón isolate presented the lowest radial growth of 0.9
the interaction between both factors was not significant. mm at 48 hours (Figure 6).
Radial growth ranged from 0.9 mm to 2.5 mm every 48
Figure 6: Radial growth (mm) of Lecanicillium spp. isolates at different times on sabouraud dextrose agar
medium
Viability of conidia at different times indicated that there were significant

For the variable viability of conidia of differences between the different isolates at 16
Lecanicillium spp isolates ranged from 71.4% to 99.7%. (p<0.0001), 18 (p<0.0001) and 22 hours (p<0.0001).
The highest viability was recorded in the Majada isolate The average percentage of viability at 16 hours was
at 22 hours after the test was established. The analysis 83%, with the Majada isolate presenting the highest
of variance carried out for conidia viability determined percentage of viability with 89%, while the Jinotega
significant differences between the different hours of isolate presented the lowest viability with 71%. At 18
evaluation (p<0.0001), between isolates (p<0.001), hours, the average viability percentage was 93%. The
likewise the interaction hour*isolate was significant San Ramón isolate presented the highest viability of
(p<0. 0001), which indicates that the viability of the 98%, while the Mankotal isolate presented the lowest
fungus isolates depends on the hour in which the viability percentage of 87%; the average viability
readings were taken, that the isolate that presented the percentage at 22 hours was 96%. The Majada isolate
highest viability of conidia at a certain hour was not the was the one that presented the highest percentage of
same at another hour, therefore we proceeded to viability with 100%. Most of the isolates presented a
perform an analysis of variance for each evaluation time high germination percentage of more than 95% at 22
to determine the behavior of the isolates. hours (Table 1).
Sampling periods
The analysis of variance performed for the data
Table 1: Percent viability of conidia of Lecanicillium spp. isolates on three sampling dates using Tukey's test
Isolates of Lecancillium spp Sampling periods
16 hours 18 hours 22 hours
Majada 89 a 95 a 100 a
San Ramón 89 a 98 a 97 a
La gotera 89 a 94 a 98 a
El Granadillo 87 a 93 a 98 a
Mankotal 76 b 87 b 92 b
Jinotega 71 b 90 a 91 c
Average 83 93 96
Means with equal letters, in the same column, do not differ significantly according to Tukey (p≤0.05).
Mass Production isolate.

In general, the results of this study showed that
the organic substrates based on soybean and rice were Performance of Lecanicillium spp. isolates on
the ones that presented the best results compared to the soybean-based substrate
sorghum-based substrate. All native isolates of All isolates of Lecanicillium spp. achieved
Lecanicillium spp produced conidia, but with different highly variable growth and sporulation on soybean-
results, some substrates favored some isolates in the based substrate, some isolates were favored in
multiplication of the fungus, while those same isolates multiplication. The analysis of variance determined
were not favored in other organic substrates. The significant differences in the yield in the number of
analysis of the yield in number of conidia per gram of conidia per gram, which indicates that each isolate has
substrate indicated that there were significant certain very particular characteristics in its
differences between substrates (p<0.0001), between multiplication (p<0.0002). The isolate La Gotera
isolates (p<0.0039) and the interaction isolate*substrate presented the highest yield with 1.08E+09 conidia per
was significant (p<0.0001), which indicates that the gram on substrate, while the isolate Mankotal presented
type of substrate influences in different ways on the the lowest yield with 1.90E+08 conidia per gram. The
multiplication of the fungus of each isolate. Due to isolates Majada, Jinotega, Granadillo and San Ramón
these results we proceeded to perform an analysis of presented similar intermediate yields between 7.42E+08
variance for each substrate with each of the isolates to and 4.21E+08 conidia per gram of substrate (Figure 7).
determine the performance in terms of yields of each
Figure 7: Average yield of native isolates of Lecanicillium spp. after 20 days of incubation on the soybean-based
organic substrate. soybean-based organic substrate error
Performance of Lecanicillium spp. isolates on rice (p:0.0337), which indicates that each isolate has certain
substrate very particular characteristics in multiplication. All the
All native isolates of Lecanicillium spp were isolates presented very variable yields. The San Ramón
able to grow, but with different yields, some substrates isolate presented the highest yield of 5.95E+08 conidia
favored some isolates in the multiplication of the per gram on substrate, the Jinotega, Majada and
fungus, while those same isolates were not favored in Mankotal isolates were statistically like the San Ramón
another organic substrate. The analysis of variance isolate, while the El Granadillo and La Gotera isolates
determined significant differences in the yield in the presented lower yields of 2.35E+08 and 2.14E+08
number of conidia per gram on rice-based substrate conidia per gram (Figure 8).
Figure 8: Average yield of native isolates of Lecanicillium spp. after 20 days incubation on the rice-based organic
substrate. Lines above the bars indicate the standard error
Performance of Lecanicillium spp. isolates on substrate, some isolates were favored in the
sorghum-based substrate multiplication of some isolates, while those same
In general, all isolates achieved highly variable isolates were not favored on other organic substrate
growth and sporulation on sorghum-based organic evaluated. The analysis of variance indicates that there
are significant differences among the isolates evaluated isolates La Gotera, Jinotega and El Granadillo presented
(p:0.001). The isolates with the highest yields were yields below the average production of the isolates
Mankotal 3.29E+08 and San Ramón 2.83E+08, while evaluated with values of 6.90E+07, 6.60E+07 and
the Majada isolate presented an intermediate yield of 5.70E+07, respectively (Figure 9).
1.74E+08 conidia per gram of substrate, while the
Figure 9: Average yield of native isolates of Lecanicillium spp. after 20 days incubation on sorghum-based
substrate. Lines above the bars indicate the standard error
Parasitism of Lecanicillium spp under In Vitro isolate presents different degrees of parasitism on the
conditions uredospores. The percentage of parasitism ranged from
All isolates caused parasitism to H. vastatrix 16.6% to 2.36%, with the Jinotega isolate showing the
uredospores except for the Majada isolate and the highest percentage of parasitism. The isolates La
control for the duration of the trial. The analysis of Gotera, El Granadillo and San Ramón were statistically
variance performed for In Vitro parasitism of different like the Jinotega isolate, while the Mankotal isolate
isolates of Lecanicillium spp on the uredospores of H. presented the lowest percentage of parasitism of 2.36%,
vastatrix, determined significant statistical differences and the control isolate did not present parasitism as
among the isolates (p:0.0014), indicating that each expected (Table 2).
Tabla 2: Parasitismo In Vitro de aislados nativos de Lecanicillium sp sobre uredosporas de H. vastatrix

Isolates Percentage of parasitism Categories Tukey (0.05)
Jinotega 16.61 A
La Gotera 15.37 AB
El Granadillo 6.39 ABC
San Ramón 2.74 ABC
Mankotal 2.36 BC
Majada 0.00 C
Testigo 0.00 C
Means with letters in common within the same column did not differ significantly according to Tukey (p≤0.05).
The isolate Majada and the control did not any uredospores, it was perhaps because it failed to find
present parasitism during the bioassay evaluation time. any uredospores in its growth trajectory as observed in
In the case of the Majada isolate that failed to parasitize the follow-up at the microscopic level.
Figure 10: In Vitro parasitism test. Ungerminated spore of H. vastatrix (A). Structures of Lecanicillium spp
verticillate conidiophores emerging from uredospores of H. vastatrix (B). Germinated conidia of Lecanicillium spp
(C). Interaction between Lecanicillium spp and H. vastatrix disintegrating uredospores (D)
In Vitro parasitism of Lecanicillium spp on H. analysis of variance for In Vitro parasitism of

vastatrix pustules Lecanicillium sp on H. vastatrix uredospores on leaves
The percentage of parasitism caused by native found significant differences among the treatments
isolates of Lecanicillium spp on leaf rust pustules under evaluated (p:0.0001), all isolates caused parasitism on
laboratory conditions ranged from 88.33 to 66.43 at 72 H. vastatrix pustules. However, no statistical
hours after establishing the test. No parasitism was differences were found within the isolates evaluated
observed in the control during this same evaluation during the evaluation period, all isolates behaved in a
period (Table 3). The Kruskal Wallis non-parametric similar way on leaf parasitism (Table 3).
Table 3: Comparative means of In Vitro parasitism of native isolates of Lecanicillium sp on pustules of H. vastatrix
Isolated Parasitism % Categories
Testigo 0.00 A
Mankotal 66.43 B
Majada 70.08 B
San Ramón 71.95 B
Jinotega 78.75 B
El Granadillo 86.48 B
La Gotera 88.33 B
Means with letters in common, within the same column do not differ significantly according to Tukey(p≤0.05).
DISCUSSION
The results of our study with respect to The physiological characteristics were like the
microscopic morphometric characterization were studies carried out by González (2001), who describes
similar to those of Icochea (2004), describing erect that Lecanicillium spp. presents creamy white colonies,
phialides, wide at the base and ending in a thin tip from with striations on the back and cottony colonies, this is
which the conidia emerge, generally in groups of two to a characteristic of this fungus which produces a
six, these conidiogenous cells measure 11 to 30 µ in compacting in the whole colony that makes it difficult
length x l.5 to 2 µ in diameter, the conidia are to detach its mycelium. In our study the wrinkling of
ellipsoidal from 2 to 4 µ x 1 to 1.5 µ. The phialides are the fungus was observed mostly in the SDA and EMA
solitary or in whorls originating from straight culture media. In the PDA culture medium, the growth
conidiophores. Likewise, Zare and Gams (2001) of the colonies of the isolates presented the same off-
describe relatively short conidiogenous conidiogenous white coloration, but showed little vertical growth and
cell phialides 11 to 30 µ x 1.4 to 1.8µ, agglomerated little cottony, in addition, a ringed growth of concentric
and strongly tapered, produced singly or in groups of up spaced circles was observed, since the hyphae seek a
to 6 directly on prostrate hyphae, or on short, erect better use of nutrients, adhesion and dissemination on
conidiophores sometimes also produced secondarily on the nutrient medium (Argueta, 2011). The radial growth
earlier phialides. Conidia formed on heads at the apex of native isolates of Lecanicillium spp. is like studies
of the phialides, typically short ellipsoidal in shape reported by Zare and Gams (2001), who describe that
ranging from 2.5 to 4.2 µ x 1 to 1.5 µ. this fungus presents a colony growth that reaches 15 to
25 mm in diameter in 10 days at 24°C in PDA culture nutritional values of the culture media. The growth
medium. It is important to mention that all isolates of dynamics of Lecanicillium spp. isolates in PDA
Lecanicillium spp presented cottony colonies, but not medium showed that most of the isolates presented a
powdery colonies, which were observed in the three similar growth pattern over time with less variability
media evaluated (Figure 2). compared to that found in the EMA culture medium;
however, radial growth in PDA culture medium was
The radial growth of native Lecanicillium spp higher in most of the sampling periods. The PDA
isolates at different times for the SDA culture medium culture medium has a pH of 5.6 ± 0.2 which could
was less variable (Figure 6) compared to PDA and allow the isolates to have a radial growth to be favored
EMA culture media. It is important to emphasize that in and present a uniform growth. The growth dynamics of
SDA culture medium the growths were quite uniform the isolates on SDA culture medium was more regular
compared to those obtained in EMA and PDA. (2007) with the highest growth of all isolates at 336 hours
cited by Retamal (2008) indicate that the culture (Figure 6).
medium in which L. lecanii grows and sporulates best is
Sabouraud Dextrose Agar, which is one of the most Viability Most of the isolates presented a
widely used culture media by insect pathologists. germination close to 100% (Table 1), with adequate
characteristics for mass production in terms of viability
The radial growth of Lecanicillium spp. since this parameter is determinant for mass
isolates in the EMA culture medium was lower reproduction according to Monzon (2001). The results
compared to the other culture media; this medium has a obtained in the test of viability of conidia of different
pH of 4.7 ± 0.2, which could be unfavorable for isolates of Lecanicillium spp, show that in the majority
mycelial growth (Figure 4). Pereira et al., (2007) affirm the viability of conidia has a tendency to increase as the
that the variation of pH in the culture medium is a hours pass, on each date the isolates presented a
determining factor in the behavior of fungal species. variability in viability from one date to another; that is,
Studies by López and Carbonell (1999) confirm these those that were better at 18 hours were not necessarily
findings where V. lecanii grows better at pH close to 7 better at 22 hours in their germination, a rapid
(Figure 4). Espinosa and Vallejos (2016) comparing germination helps to decrease the time of exposure to
different culture media, found that the highest average environmental conditions and achieve a rapid effect on
radial growth rate of Beauveria bassiana was in PDA the pest (Malpartida et al., 2013). The effectiveness of
culture medium; in our study the same particularity was the fungus in the field depends on its ability to colonize
found when comparing different culture media with a substrate and infect its host, which in turn is
different native isolates of Lecanicillium spp finding determined by its viability (Monzon, 2001). For this
that the highest growth was recorded in PDA culture reason, the determination of this characteristic is
medium (Figure 5). Some culture media could stimulate important in the selection of strains for biological
fungal growth and influence some physiological control; the higher the viability in a shorter time under
characteristics such as viability and sporulation. Pereira laboratory conditions, it is presumed that it will present
et al., (2007) indicate that the variation of pH in the better adaptability to environmental conditions and
culture medium is determinant in the behavior of fungal better effectiveness on the host; also, Steinkraus (2006)
species. The demand for a particular nutrient often suggests that viability is related to the ability to spread
varies not only within a single species, but also for within a host population and determines its potential as
individual strains of a species (Iskandarov et al., 2004). a microbial control agent.
The growth dynamics of Lecanicillium spp. Spore yield in organic substrates the general
isolates on PDA medium in the last three evaluation analysis shows that the isolate La Gotera stands out
periods showed no statistical differences in the radial above the others in the soybean-based substrate,
growth of the isolates, and it is possible that they are possibly there is some specificity between the
entering a period of low metabolism, which could allow vegetative material and genetic characteristics of the
the fungus to develop survival strategies. Milner et al., isolate, this substrate provides better nutritional
(1991) affirms that it is possible that some isolates enter conditions in relation to carbon / nitrogen and other
a type of physiological dormancy, which would allow essential elements for its multiplication (Figure 7). For
them to survive for prolonged periods in the field the rice-based substrate, the isolates that presented the
outside the host and give them better adaptability best yields were San Ramón and Jinotega (Figure 8). In
characteristics and better performance as biological this study, it was observed that this rice substrate
controllers. All isolates showed the same growth pattern absorbs moisture efficiently in the process, and presents
over time. At 480 hours, most of the isolates on SDA adequate aeration; however, it was observed that the
medium showed growth rates of less than 1.3 mm growth in the substrate grows in a compact manner that
(Figure 6). Radial growth in this culture medium was hinders the separation of the conidia and substantially
lower on most of the dates evaluated compared to those affects its yield. In some fungi such as Lecanicillium
obtained in PDA; radial growth is determined by lecanii, the amount of conidia can decrease due to the
several factors such as: pH, N/C ratio, as well as other high compaction exerted by the fungus on the rice,
which prevents internal aeration and reduces conidial uredospores showed different degrees of parasitism
production (Cortez, 2007). There are very important (Table 2). Gonzalez (2001) obtained similar results
characteristics to use a certain substrate for mass where he found different degrees of parasitism on
reproduction, one of them is that it should have easy uredospores of H. vastatrix. Parasitism In Vitro was
separation of conidia at the time of harvest, and another evidenced 72 hours after inoculation of Lecanicillium
is that the substrate should have good consistency spp. conidia on rust uredospores, destruction of the cell
throughout the process of mass reproduction. In the wall of the uredospores could be observed through the
sorghum-based substrate, the isolates that were favored light microscope (Figure 10). This same phenomenon of
in spore production were Mankotal and San Ramón inhibition of germination of uredospores of H. vastatrix
(Figure 10). occurred in a study by Eskes (1987) who determined
that in addition to being a mycoparasite of H. vastatrix,
To consider a substrate as suitable for mass it was also an inhibitor of uredospore germination.
production, it must stimulate a high concentration of
conidia (Cortez, 2007). Similarly, Roberts and Yendol The percentages of parasitism on rust pustules
(1971) argue that a substrate with good attributes to of native isolates of Lecanicillium spp are high in this
produce conidia of a strain of an entomopathogenic study, the values fluctuated between 88.33 and 66.43%
fungus should be considered low cost and easy to (Table 3). Gonzalez (2001) found similar parasitism
acquire. In our study, this soybean substrate, although percentages on H. vastatrix sori and reduced uredospore
good conidia production and low cost were obtained, germination to 0% and reached 81.03% parasitism. In
does not have a good capacity to absorb moisture in the this study, parasitism on pustules was quantified by
preparation of the substrate or in the incubation period, visual observation and a pustule with whitish mycelium
which makes it difficult to handle, and for this reason was parasitized. Mahfud et al., (2006) point out that the
this substrate is very susceptible to bacterial effects of some species of this genus produce
contamination. discoloration of uredospores, formation of white
mycelium on them or necrosis, depending on the time
Cortez (2007) affirms that conidial production of evaluation. The findings found in this study under
varies according to the substrate used for its laboratory conditions could be very useful and of great
reproduction, and that the fungus L. lecanii produces a importance. The collected isolates cause parasitism to
greater quantity of conidia when cultivated in substrates the pathogen, and it was also demonstrated that the
that allow greater aeration; however, in the results isolates present different degrees of parasitism on the
obtained in this study it was observed that to obtain uredospores of Hemlieia vastatrix. These results could
greater production of conidia it is also necessary for the motivate others to develop effective and inexpensive
substrate to have good nutritional values. For this methods for mass reproduction, and their potential use
reason, we can say that the nutritional values of a for biological control of coffee rust should be
substrate are a determining factor in obtaining a greater considered by incorporating disease management
number of conidia. Volcy and Pardo (1994), who strategies under field conditions.
mention that a good substrate to produce conidia must
have a high content of carbohydrates, nitrogen, CONCLUSION
microelements, B complex vitamins and a high Six native isolates of the genus Lecanicillium
concentration of ions necessary for growth and spp. were obtained, the microscopic and macroscopic
sporulation. characteristics were similar in the three-culture media.
The growth rate is influenced by the artificial culture
Aceves (2008) by means of a proximal medium and by the type of isolate. The viability of
chemical analysis of different organic substrates for conidia of native isolates of Lecanicillium spp. depends
mass reproduction showed that there is a correlation on the isolate and increases with time; the best viability
between nutritional values and spore production. reading time was at 22 hours, since most of the spores
Figueroa et al., (2007) argue that sporulation are already germinated, and mycelium production
performance on a substrate can also be influenced by makes the reading more accurate. The performance of
factors such as culture technique, initial inoculum Lecanicillium spp. isolates on different organic
concentration, temperature, humidity, aeration, and substrates is determined by a good initial inoculum,
incubation time. In our study it was also observed that good aeration of the substrate, adequate multiplication
this substrate presented a deficiency in terms of technique, as well as the ratio of nitrogen and carbon
moisture absorption during the production process, content. In the In Vitro parasitism tests, the Jinotega and
which generates a longer drying time, being somewhat La Gotera isolates showed the highest percentages of
tedious for its use in the mass reproduction of fungi, for parasitism on uredospores and rust pustules,
such reasons the use of this substrate is not respectively, indicating that the level of parasitism is
recommended for such purposes, and because there is a also determined by the type of isolate.
potential risk of bacterial contamination as observed.
The parasitism of Lecanicillium spp. on

ACKNOWLEDGMENTS (CETREX). Consultado 08-05-2020. Disponible en

The authors of this research thank the https://www.cetrex.gob.ni/website.
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support of this study. (= Verticillium) lecanii en diferentes sustratos y
patogenicidad. Agricultura técnica en México,
33(1), 83-87.
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Scholars Journal of Agriculture and Veterinary Sciences

Abbreviated Key Title: Sch J Agric Vet Sci

In Vitro Evaluation of Native Isolates of Lecanicillium spp (Berk &

DOI: 10.36347/sjavs.2023.v10i08.001 | Received: 09.07.2023 | Accepted: 02.08.2023 | Published: 11.08.2023

Abstract Original Research Article

INTRODUCTION 2004). Pest control in coffee growing has been using

Microscopic characterization Viability of conidia

Mass Production manually homogenized to ensure uniform growth of the

RESULTS Microscopic characteristics

Viability of conidia at different times indicated that there were significant

Mass Production isolate.

Tabla 2: Parasitismo In Vitro de aislados nativos de Lecanicillium sp sobre uredosporas de H. vastatrix

In Vitro parasitism of Lecanicillium spp on H. analysis of variance for In Vitro parasitism of

The parasitism of Lecanicillium spp. on

ACKNOWLEDGMENTS (CETREX). Consultado 08-05-2020. Disponible en

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