Ciencias
Agrícolas y Veterinarias
10.57201/IEUNA2313332
Sección: Artículos Originales
*Autor correspondiente:
claudiagonzalez@cyt.uni.edu.py
Editor de área:
Andrea
Alejandra Arrúa Alvarenga
, Universidad
Nacional de Asunción.
Editor invitado:
Guillermo
Enciso, Centro
de Desarrollo e Innovación Tecnológica (CEDIT)
Recibido:
16
de febrero de 2023
Aceptado:
19
de junio de 2023
Recibido
en versión modificada:
22
de junio de 2023
Este
es un artículo publicado en acceso abierto bajo una licencia Creative Commons “CC BY 4.0”. 
Declaración de conflicto: Los autores
declaran no tener conflicto de interes.
e-ISSN
2709 -0817
Como citar: González, C. E. , Gayoso, D. S.,
Salinas, A., Bich, G. A. & Castillo, M. L. (2023). Primer relevamiento de
hongos aislados de suelos e insectos plagas asociados a cultivos orgánicos de
cáñamo industrial (Cannabis sativa L.) en Paraguay.Revista investigaciones y estudios
– UNA, 14(1), 104-109.
First survey of First survey fungi
isolated from soils and pest insects associated with organic plantations of
industrial hemp (Cannabis sativa L.) in Paraguay
Primer relevamiento de hongos aislados de suelos e
insectos plagas asociados a cultivos orgánicos de cáñamo industrial (Cannabis
sativa L.) en Paraguay
Claudia Elena González1*
,
David Santiago Gayoso1 , Alan
Salinas2 ,
Gustavo A. Bich3 ,
María Lorena Castrillo3
Abstract.
The cultivation of Cannabis sativa has gained
importance in recent years in Paraguay and other Latin American countries where
legal regulations allow its cultivation for medicinal use, as fiber, and also
as food. Since several companies apply the organic cultivation model to
guarantee quality and certify their product, it is necessary to study
biological control alternatives that guarantee sustainable agriculture. In this
study, microorganisms present in the soil of Cannabis sativa cultivation
and those that infected pests found on the plant were isolated and identified.
The serial dilution method was used to isolate microorganisms from the soil and
direct seeding on specific culture media to isolate them from the bodies of
pest insects. The isolated genera from the soil were Rhizopus, Aspergillus,
Absidia, Alternaria, Mucor, Penicillium and Fusarium;
the isolated genera from pest insects were Cladosporium, Alternaria,
Aspergillus, and Fusarium. This study is the first report on fungi
in soils and pest insects in industrial hemp cultivation in Paraguay.
Keywords: agroecosystem,
management, organic, Paraguay.
Resumen. El
cultivo de Cannabis sativa ha tomado una gran importancia en los últimos
años en Paraguay y en otros países de América Latina donde las reglamentaciones
legales permiten su cultivo para usos medicinales, como fibra y también como
alimento. Debido a que varias empresas aplican el modelo de cultivo orgánico
para garantizar la calidad y además para llegar a certificar su producto es
necesario estudiar alternativas de control biológico que garanticen una
agricultura sostenible. En este estudio se aislaron e identificaron los
microorganismos presentes en el suelo de cultivo de Cannabis sativa y
también aquellos que infectaron a los insectos plagas encontrados sobre la
planta. Se utilizó el método de dilución seriada para aislar microrganismos del
suelo y para aislarlos de los cuerpos de insectos plagas la siembra directa en
medios de cultivos específicos. Los géneros aislados del suelo fueron Rhizopus, Aspergillus, Absidia,
Alternaria, Mucor, Penicillium y Fusarium; los
géneros aislados de insectos plaga fueron Cladosporium,
Alternaria, Aspergillus y Fusarium. Este es la primera
descripción sobre la presencia de hongos en suelos e insectos plagas en cultivo
de cáñamo industrial en Paraguay
Palabras
clave: agroecosistema, manejo, orgánico,
Paraguay.
Introduction
In recent years, non-psychoactive industrial hemp (Cannabis
sativa L.) has experienced significant growth due to its usefulness in the
textile, medicinal, and food industries (Rupasinghe et al., 2020). The cultivation of hemp in
Paraguay has increased eight times, ranging from 600 ha in 2019 to 5000 ha in
2022 (Prohibition Partner, 2022). The reasons include its decriminalization and
high demand in domestic and international markets (Reuters,
2023). The rapid expansion of organic crops has increased focus on pest
management research. Nevertheless, limited information on the subject presents
significant challenges to producers in effectively managing diseases caused by
fungal and insect pests, which emerge as a result of high crop density and
specific management practices implemented in the field (Sunoj Valiaparambil et
al., 2023; Zheljazkov et al., 2023). Soil is considered one of the main sources
of inoculum of phytopathogens (Johns et al., 2021).
The phytopathogen Fusarium oxysporum has been found in soils of C.
sativa crops in North America, Israel, and Italy, raising concerns among
hemp growers worldwide due to the significant commercial losses. This pathogen
can infect the plant from its seedling stage and spread through infected mother
plants, generating varying degrees of aggressiveness depending on the
haplotypes (Jerushalmi et al., 2022). Previous studies in C. sativa have
revealed the presence of 12 haplotypes of Fusarium cannabis, of which
five have been reported to cause significant economic losses due to plant
diseases and mycotoxin residues (Gwinn et al.,
2022). Hemp crops are also vulnerable to pest, such as the red spider mite (Tetranychus
urticae), which can significantly reduce production and commercial product
quality. To control this pest growers, use mites from the genera Neoseiulus
californicus and Phytoseiulus persimilis as biological control
agents. In addition, hemp farmers explored the potential of entomopathogenic
fungi, whose effect as bioinsecticides is well-known in other crops (Jimenez
Lara, 2022). In 2019 in Ecuador, Tetranychus urticae, caused 60- 80%
losses in strawberry crops, which motivated the evaluation of the
entomopathogenic activity of Bacillus subtilis in vitro (Mendoza-Léon et
al., 2019). While the effects of fungi such as Beauveria bassiana and Metarhizium
anisopliae as biocontrol agents are well-known, bacteria have been little
studied. The results obtained were very encouraging and suggest that this
technique could be used as a biocontrol alternative for other crops. (Pacheco
Hernández et al., 2019). In Paraguay, during research looking to obtain
biological control agents from the compost used to grow Cannabis
seedlings, several genera of filamentous fungi were isolated and molecularly
identified as Aspergillus sp., Penicillium sp., Cladosporium
sp., Geomyces sp., Plectosphaerella sp., and Rasamsonia
sp. These genera are common in crop soils, although Cladosporium sp.
should be considered a potential threat in C. sativa as it is a pathogen
in other crops (Álvarez Beauchamp, 2022). This research aimed to isolate and
identify microscopic fungi associated with the soils and insects of organic
non-psychoactive industrial hemp crops on a farm in the Eastern Region of
Paraguay to characterize the diversity.
Materials
and methods
Sampling site
The sampling places were selected
according to farmers that develop organic crops and previous investigations
agreements. Samples were collected at nine georeferenced points selected
randomly in the certified organic soils belonging to EVONA S.A. (Figure 1). The
production fields were located in Hernandarias city, 7.32 km from route PY07 Km
28 (25°18'7"S 54°37'54"W). Georeferencing was performed using the
Android operating system application GPS Fields Area Measure PRO.
Figure
1.
Sampling site of hemp plantation. A) Georeferenced sampling points,
Hernandarias, Paraguay. B) Types of soils. C) Detail of parcel of sampling. D)
Selected hem plantation.
Soil samples
According to data published by the
Departamento de Planificación Ambiental del Ministerio de Agricultura
(Paraguay), the soil type belongs to the Oxisol classification, soils derived
from basalt that show an undulating profile in the form of hills followed by
flat areas, the content of mica, apatites, feldspars and ferromagnesian
minerals must be below 10% since they are weatherable.
Subsequently, soil samples weighing
200 grams each, 1800 grams for all, were extracted for pH determination and
microbiological analysis. The soil samples came from places where hemp was grown
between April and August 2022, in plots where hemp cultivation is anticipated
in the future, and from residual substrate. The sample preparation followed the
methodology proposed by Jerke et al., (2019). The soil samples were collected
in magnetic-closure bags for transport to the Ciencia y Tecnología laboratory
at the Universidad Nacional de Itapúa.
Solutions were prepared in sterile
distilled water from nine soil samples to measure pH. Twenty grams of each
sample was weighed and added to a beaker containing 50 ml of distilled water.
The mixture was stirred with a glass rod for 2 minutes and left to settle for
15 minutes. The pH of the supernatant was measured using a QUIMIS Q400AS
240V-10W benchtop pH meter. To isolate fungi from the
soil samples prepared for hemp cultivation, serial dilutions of 10-1,
10-2, and 10-3 were prepared in sterile distilled water.
They were then seeded onto nutrient agar plates containing chloramphenicol
antibiotic (150 mg/l). The plates were labeled and incubated at 25°C for 5 to 7
days until colony growth was observed (Gonzalez et al., 2013; Humber, 1997).
Insect samples
All dead insects found on the plants or
laying on the soil were collected. The insect samples were transported sterile
jars to the Ciencia y Tecnología laboratory (Universidad Nacional Itapúa). They
were processed using the wet chamber technique and the direct puncture
technique of the soft tissues, which were then cultured on potato dextrose agar
(PDA) agar plates. The insects were identified in the field through
morphological features.
Fungal colonies were observed both macroscopically and
microscopically. For microscopic observations, a compound microscope (Zeiss
Primo Stard Trinocular®) with an incorporated digital camera was
used to capture images at magnifications of 400X and 1000X. The taxonomic guide
of Dr. Humber (1997) was used for the microscopic identification of the fungal
genus. The fungal colonies were observed in the Faculty of Science and
Technology laboratory at UNI.
Results and Discussion
A total of nine samples of oxisol soils and 12 samples of
insects were collected from the hemp plantations (Figure 2). The main insects
genera found were Diabrotica, Empoasca, Naupactus and Autographa (personal
communication with Ing. Alan Salinas, EVONA S.A.).
Figure 2. Examples of
organisms-inhabiting in hemp plantations sampled in Paraguay. A and B fungal
colonies isolated from soil samples. C and D insects collected from hem
plantations.
Of
the 42 fungal colonies growing in the Petri dishes with PDA from soil samples,
30 were confirmed through microscopic examination to represent one of the seven
genera: a Rhizopus, Aspergillus, Absidia, Alternaria, Mucor,
Penicillium, and Fusarium. In all the oxisol soils from the hemp
plantation the genera Fusarium, Aspergillus, Penicillum
and Cladosporium were found. The absence of entomopathogenic fungi of
the species B. bassiana and M. anisopliae may be attributed to
the acidic conditions of the soil and the high temperatures encountered at the
various sampling sites. From the collected insects Cladosporium, Alternaria,
Aspergillus, and Fusarium were isolated. In our study, all the
samples were collected during the continuous dry season prevailing in our
region, with a remarkably absence of regular rainfalls, which may alter the
occurrence of many groups of microorganisms from samples of soil. It has been
suggested that the presence of B. bassiana to be very low during periods
of scarce rainfall and intense drought, increasing during rainy months with
higher humidity and temperatures of 20-30°C, conditions favorable for spore
dispersal and germination. However, the genus M. anisopliae was found to
invade tissues of dead insects when temperatures reached 40°C. These data
highlight the importance of climatic conditions in the development of some
pathogens and emphasize the need to consider them in future studies of
pathogenicity related to hemp cultivation (García-García et al., 2011). Another
important aspect to consider is that the pH in the analyzed soils was rather
acid, varying between 5.2 and 5.71. The field frequency of entomopathogenic
fungi was low, which could be related to crop management, environmental
conditions, availability of susceptible hosts, pathogen virulence, and host
crop characteristics, among others (Borges, 2007;
Vänninen, 1996). Climatic factors such as moderate temperatures, abundant
rainfalls, and high humidity are correlated with the occurrence of
entomopathogenic fungi (Clifton et al., 2015; Jaronski, 2010; Yaginuma, 2007).
Similarly, Silva Guerra et al., (2009) reported
the effect of moderated temperatures and high percentajes of humidity favours
the persistence of conidia in soil, where M. anisopliae was found to be
more resistant than M. acridum . The presence of Penicillum,
Alternaria, Aspergillus, and Fusarium genera in soil and pest insect
tissues would merit further study of their pathogenicity under controlled
laboratory conditions to deepen knowledge of their potential use as biocontrol
agents of hemp crop pests.
Conclusions
This work has made it possible to know
the fungal diversity present in soils and pest insects associated with organic
crops of industrial hemp (Cannabis sativa, L.) for the first time in Hernandarias,
Alto Paraná, Paraguay. All of the isolated and identified fungi of the genera Alternaria,
Rhizopus, Absidia, Mucor, Penicillium, Aspergillus,
Fusarium, and Cladosporium have been stored in the InBioMis
culture collection, Misiones Argentina, and the Faculty of Science and
Technology of the National University of Itapúa for future molecular
identification assays.
The present work allowed us to establish
links between the National University of Itapúa, the Institute of Biotechnology
of Misiones, Argentina, and the company Evona S.A. so that we can continue
researching and interacting in the future for the benefit of organic production
of industrial hemp.
Contribución de los autores
Conceptualization: C.E.G., G.A.B., M.L.C. Experiment design: C.E.G.,
D.S.G, G.A.B., M.L.C. Experiment
execution: C.E.G.,
D.S.G., A.S. Experiment verification: C.E.G., D.S.G Data analysis/interpretation: C.E.G., D.S.G, G.A.B., M.L.C. Statistical analysis: Manuscript preparation: C.E.G.,
G.A.B.Manuscript editing and revision: C.E.G., G.A.B., M.L.C. Approval of the final manuscript version: C.E.G.,
G.A.B., M.L.C.
Fuente
de Financiamiento. Sin financiamiento externo.
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