Betsabe Leon Ttacca

Almendra Astete Farfán

Luz Leonor Mattos Calderón

Enrique Arévalo Gardini

Rev. Fac. Agron. (LUZ). 2022, 39(1): e223922

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n1.22

Crop Production

Associate editor: Professor Beltran Briceño

Keywords:

Fungus

Blueberry

Colonization

Identication

Strains

Endophytic mycobiota associated

to plants of

Vaccinium corymbosum L. in Cañete valley- Perú

Micobiota endófita asociada a plantas de Vaccinium corymbosum L. en el valle Cañete- Perú

Micobiota endofítica associada a plantas de Vaccinium corymbosum L. no vale do Cañete - Perú

Escuela profesional de Agronomía, Facultad de Ciencias

Agrarias, Universidad Nacional de Cañete. Jr. San Agustín

124, San Vicente de Cañete Lima, Perú.

Laboratorio de Sanidad Vegetal, Facultad de Ciencias

Agrarias, Universidad Nacional de Cañete. Jr. San Agustín

124, San Vicente de Cañete Lima, Perú.

Escuela Profesional de Agronomía, Departamento

Académico de Fitopatología, Universidad Nacional Agraria

La Molina, Apartado postal 12-056 - La Molina / Lima-Perú.

Universidad Nacional Autónoma del Alto Amazonas,

Yurimaguas, Loreto, Perú. Instituto de Cultivos Tropicales,

Taparopo, San Martín, Perú.

Received: 12-05-2021

Accepted: 01-12-2021

Published: 04-03-2022

Abstract

Endophytic fungi (EF) are microorganisms that live asymptomatically

within plant tissues. They can confer benets to the plant as inducers of

resistance to pests and diseases, tolerance to abiotic factors, plant growth

promoters and mycoparasites of pathogens. This study aimed to isolate,

identify, and determine occurrence frequency of endophytic fungi in

stems and leaves of blueberry plants collected from ten farms in Lima

and Ica Region, Peru. Productive branches of 39 healthy plants were

sampled. For the isolation of EF, 702 tissue sections were disinfected and

transferred to Petri dishes with potato dextrose agar (PDA) medium, and

reproductive structures were induced for identication. A total of 134 EF

strains were isolated successfully. Nine genera were identied (Trichoderma,

Aspergillus, Alternaria, Ulocladium, Cladosporium, Fusarium, Nigrospora,

Lasiodiplodia and Stemphylium). EF occurrence frequency varied greatly

between organs, being in leaves more frequent than the stems; likewise, the

genera Alternaria and Cladosporium were the most frequent and with most

colonized in plant tissues, while Aspergillus, Ulocladium and Lasiodiplodia

were the least frequent. These strains could be useful to control plant diseases

and, therefore, of great interest in sustainable agriculture.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2022, 39(1): e223922. January - March. ISSN 2477-9407.

2-7 |

Resumen

Los hongos endótos (HE) son microorganismos que viven

de manera asintomática dentro de los tejidos vegetales. Pueden

conferir benecios a la planta, comoinductores de resistencia a

plagas y enfermedades, tolerancia a factores abióticos, promotores

de crecimiento vegetal y micoparásitos de patógenos. El objetivo

del presente trabajo fue aislar e identicar los hongos endótos,para

determinar su frecuencia de aparición en tallos y hojas de plantas de

arándano,colectadas en 10 fundosde la región Lima e Ica, Perú. Se

muestrearon ramas productivas de 39 plantas sanas, y fueron llevadas

al laboratorio de Sanidad Vegetal de la Universidad Nacional de

Cañete. Para el aislamiento de HE, 702 secciones de tejidos fueron

desinfestados y transferidos a placas Petri con medio de cultivo papa

dextrosa agar (PDA) y se indujo estructuras de reproducción para

la identicación. Un total de 134 cepas de HE fueron aisladas, de

los cuales se identicaron nueve géneros (Trichoderma, Aspergillus,

Alternaria, Ulocladium, Cladosporium, Fusarium, Nigrospora,

Lasiodiplodiay Stemphylium). La frecuencia de aparición de los

HE fue muy variada según los órganos. Las hojas tuvieron mayor

frecuencia que los tallos; así mismo, los géneros Alternaria y

Cladosporium resultaron ser los más frecuentes y con mayor

colonización en los tejidos de las plantas mientras que Aspergillus,

Ulocladium y Lasiodiplodia fueron los menos frecuentes. Estas cepas

podrían ser útiles para el control de enfermedades vegetales y, por

consiguiente, de gran interés en la agricultura sostenible.

Palabras clave: Hongo, arándano, colonización, identicación,

cepas.

Resumo

Os fungos endofíticos (FE) são microrganismos que vivem

de forma assintomática nos tecidos vegetais. Eles podem conferir

benefícios à planta, como indutores de resistência a pragas e doenças,

tolerância a fatores abióticos, promotores de crescimento vegetal

e micoparasitas de patógenos. O objetivo deste trabalho foi isolar

e identicar fungos endofíticos, para determinar sua frequência de

aparecimento em caules e folhas de plantas de mirtilo, coletados

em 10 fazendas na região de Lima e Ica, Peru. Ramos produtivos

de 39 plantas sadias foram amostrados e levados ao laboratório de

Fitossanidade da Universidade Nacional de Cañete. Para o isolamento

de FE, 702 cortes de tecido vegetal foram desinfetados e transferidos

para placas de Petri contendo como meio de cultura batata dextrose

ágar (PDA), e as estruturas de reprodução foram induzidas para

identicação. Um total de 134 cepas de FE foram isoladas, das

quais nove gêneros foram identicados (Trichoderma, Aspergillus,

Alternaria, Ulocladium, Cladosporium, Fusarium, Nigrospora,

Lasiodiplodia e Stemphylium). A frequência de aparecimento de FE

foi muito variada de acordo com os órgãos. As folhas tiveram maior

frequência do que os caules; da mesma forma, os gêneros Alternaria e

Cladosporium foram os mais frequentes e com maior colonização nos

tecidos vegetais, enquanto Aspergillus, Ulocladium e Lasiodiplodia,

foram os menos frequentes. Essas cepas podem ser úteis para o

controle de doenças de plantas e,portanto, de grande interesse uma

agricultura sustentável.

Palavras-chave: Fungo, mirtilo, colonização, cepas, identicados.

Introduction

The blueberry is the second most exported product of the

Peruvian fruit and vegetable basket. The United States of America

is one of the main destinations for Peruvian exports with a 55%

share during the 2019-2020 agricultural campaign, because they are

the largest consumer of this fruit worldwide (Redagricola, 2020).

The requirements of the global blueberry market require organic

production of this fruit, with the biological control of diseases being

one of the key factors in this practice. A study of its endophytic

mycobiota would allow the identication of microorganisms that

could help in this process, contributing to the current demands of

international markets.

Endophytic fungi are symbiotic organisms that live in association

with plants for most or all of their life cycle. These microorganisms

cover different orders, they are found inside the tissues, in the

intercellular spaces and sometimes, intracellularly in leaves, stems

and owers, absorbing nutrients from the plant without producing

symptoms of any disease, on the contrary they provide protection

against biotic agents and abiotic (Busby et al., 2016; Carroll,

1988; Lugtenberg et al., 2016; Mane et al., 2018). In the process of

biocontrol of diseases, these microorganisms protect the host plant

mainly through antibiosis, parasitism and competition mechanisms,

which by inhibiting the growth of plant pathogens, result in better

plant performance (Segaran and Sathiavelu, 2019).

Currently, endophytic fungi are investigated in many crops,

due to their importance in plants (Busby et al., 2016; Mane et al.,

2018). They are found in some species of Vaccinium and are divided

into endophytic, pathogenic and ectotrophic (Miao et al., 2013). In

leaves of Vaccinium dunalianum, the existence of a high diversity

of these fungi is reported, managing to identify taxa with potential

antimicrobial activity, being a source of natural bioactive compounds

for future agroindustrial applications (Fan et al., 2020; Li et al.,

2016). However, in Vaccinium corymbosum there are no studies

on the presence of these microorganisms. The report of associated

endophytic fungi in blueberry production will be very useful for the

search for strains with potential for biocontrol of diseases in this

crop. With the isolation of endophytic fungi from plants, biologically

active agents can be produced on a large commercial scale, without

affecting ecological biodiversity, since they are easily cultivated in

the laboratory (Mane et al., 2018). For these reasons, the objective

of this research is to isolate, identify and determine the frequency of

appearance of endophytic fungi in stems and leaves of blueberries

(Vaccinium corymbosum L.) produced in the Cañete valley in Lima,

Peru.

Materials and methods

Sample Collection

During the months of January, February and March 2020, 39

healthy blueberry plants of the Biloxi variety, three years old, were

sampled in 13 production lots belonging to ten farms in four districts

of the province of Cañete, Lima Region (Nuevo Imperial, Imperial,

Roma and Quilmaná) and on a farm in the district and province of

Pisco, Ica Region under a denitive soil production system (Table

1). The climate of the province of Cañete is temperate with an

average temperature of 13 °C in winter and 28 °C in summer and an

average annual rainfall of 11.4 mm. The crop production system is

carried out in black polyethylene bags (instead of pots) containing

a substrate composed of peat and rice husks, all plantations are

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León et al. Rev. Fac. Agron. (LUZ). 2022, 39(1): e2239223-7 |

irrigated and fertilized through the system called “drip irrigation”. A

non-probabilistic - intentional or convenience sampling method was

used (Arias-Gómez et al., 2016), three blueberry plants with the best

characteristics in terms of growth and health were selected from each

production lot. For each plant, productive branches were extracted

from the lower, middle and upper third, the samples were labeled,

deposited in polypropylene bags and taken to the Plant Health

Laboratory of the Universidad Nacional de Cañete (UNDC) being

kept at 4 °C for 24 to 48 hours until processing.

Isolation of endophytic fungi

To obtain plant tissue sections, three leaves and three stem pieces

were taken from each third of the blueberry plant; For each leaf, a

section of 1 cm

was obtained between the central rib and the edge

of the leaf, and 1 cm lenth for each piece of stem, having 18 sections

per plant and 54 sections per lot (three plants). For the isolation

of endophytic fungi, the supercial disinfestation of the sections

of stems and leaves was carried out inside a biosafety cabinet, by

means of immersion; previously, the different sections were washed

with sterile distilled water (ADE), followed by 3 min in 2% sodium

hypochlorite, 1 min in 70% (v/v) alcohol, and three washes with

ADE (De Sousa Leite et al., 2013). Sterilized tissue sections were

placed on sterile paper towels to dry. The material was then cut into

small fragments (approximately 5 mm

), and a total of 702 tissue

sections (from 39 plants) were aseptically transferred to Petri dishes

(6 sections per plate) containing potato dextrose agar (PDA) culture

medium with chloramphenicol (500 mg.L

-1

) to rule out the presence

of bacteria (Lima and Cavalcanti, 2014; Mane et al., 2018). The

seeded Petri dishes were incubated in the dark at 25 °C for seven

days, after this time, the strains were puried by successive picking

of the youngest fungal mycelial edges until an axenic culture was

obtained, then they were placed under articial light for seven days to

induce the production of sexual and asexual reproductive structures

for identication. Strains that failed to form reproductive structures

were left under light for up to 30 days and were observed daily for

structures. The presence of a fungus was recorded as positive if it

was detected in an organ segment. The colonization percentage was

determined for each plant as the total number of sections colonized by

the fungus in relation to the total number of fragments per 100 (Russo

et al., 2016).

Morphological identication of endophytic fungi

The identication of the strains of endophytic fungi was carried

out at the genus level, using the conventional method based on their

morphological characteristics (Aharwal and Kumar, 2016). From

the axenic cultures of the fungi in PDA, the cultural characteristics

of the colony were observed, such as appearance, diameter, texture

and coloration, and the microscopic characteristics: type and size

of the vegetative and reproductive structures. Fragments of mycelia

were extracted from the cultures and placed on slides containing

lactophenol, the structures were visualized with an Olympus brand

binocular optical microscope, model CX23, series 7L88230 and with

the use of taxonomic keys of Barnett and Hunter (1998), Barron

(1968) and Watanabe (2002) endophytic fungal strains were identied

to the genus level.

Analysis of data

To analyze the relationship between the variables presence or

absence of each of the genera of endophytic fungi detected by organs

(leaves and stems) and production lots, the non-parametric Pearson

Chi-square test was used with the statistical program Infostatversión

2008 (Di Rienzo et al., 2008).

Results and discussion

Isolation and identication of endophytic fungi from leaves

and stems

In this study, a total of 133 strains of endophytic fungi were

isolated from healthy plant tissues of Vaccinium corymbosum L., 82

strains from leaves and 51 from stems. According to the cultural and

morphological characteristics of the fungi, nine genera belonging to

the Ascomycota division were identied: Alternaria, Aspergillus,

Cladosporium, Fusarium, Lasiodiplodia, Nigrospora, Stemphylium,

Trichoderma and Ulocladium (gure 1); There was also a group of

endophytic fungi (21 strains) that did not form reproductive structures,

which is why they were called sterile mycelium.

Table 1. Sampling locations for blueberry plants in the province of Cañete and Pisco, 2020, Peru.

Zone Lot Province District Farm South Latitude West Longitude

Altitude

(m a.s.l)

Zone 1 Lot 1 y 2 Cañete Quilmaná Blueagro 12°57’17’’ 76°21’39’’ 169

Zone 2 Lot 3 Cañete Quevo Imperial Agropec 13°00’35’’ 76°21’09’’ 153

Zone 3 Lot 4 Cañete Quilmaná Agroberries 12°58’08’’ 76°21’54’’ 138

Zone 4 Lot 5 Cañete Imperial Peruvian Prime (Huacachivato) 13°03’59’’ 76°20’24’’ 98

Zone 5 Lot 6 y 7 Cañete Nuevo Imperial Fundo a3f- Caltopia 13°03’49’’ 76°14’30’’ 333

Zone 6 Lot 8 Cañete Quilmaná Los Ángeles 12°57’16’’ 76°21’28’’ 159

Zone 7 Lot 9 Cañete Imperial Arándanos del Sur Cerro Alegre 13°02’02’’ 76°21’05’’ 137

Zone 8 Lot 10 Cañete Imperial Malvinas 13°02’02’’ 76°21’05’’ 137

Zone 9 Lot 11 Cañete Roma Agroconta 13°01’20’’ 76°20’47’’ 162

Zone 10 Lot 12 Cañete Quilmaná Agroconta – Victoria 12°59’13’’ 76°24’12’’ 110

Zone 11 Lot 13 Pisco Pisco Agroinversiones Valle y Pampa 13°48’19’’ 76°03’19’’ 279

Note. The selection of number of plots for each sampling zone is in accordance with availability of plots in the estates with plantations of V. corymbosum var. Biloxy of the

same age and crop management system.

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4-7 |

Figure 1. Genera of endophytic fungi identied in Vaccinium

corymbosum L. from the province of Cañete. Lima

Peru. A. Alternaria sp. B. Stemphylium sp. C. Trichoderma

sp. D. Cladosporium sp. E. Nigrospora sp. F. Aspergillus sp. G.

Ulocladium sp. H. Lasiodiplodia sp. I. Fusarium sp. at 100X.

Genus frequencies of endophytic fungi on leaves and stems

of blueberry plants

The genera of the endophytic fungi and the plant organs where

they are found vary according to the type of plant organ and the

production lot (p=0.0054) and are associated with the production

lots (p<0 .0001).

Of the total strains isolated from blueberry plants, 61.65%

were from leaves and 38.35% from stems. Being the genera

Alternaria, Cladosporium, Fusarium, Nigrospora, Stemphylium

and Ulocladium the most frequent in leaves; on the contrary,

Aspergillus, Cladosporium, Lasiodiplodia and Trichoderma

appeared more frequently in stems. Likewise, of the total strains

isolated and identied in stems and leaves, Alternaria and

Cladosporiumwere the most frequent genera with 44% and 23%

respectively, followed by Stemphylium and Nigrospora with 5%,

Trichoderma with 4% and Fusarium with 3 %; while the genera

Aspergillus, Lasiodiplodia and Ulocladium were the least frequent

with 1 %. In the production plots, the relative frequency varied from

3.01 to 14.29%, in plot 2 it was observed with the highest presence

of strains of endophytic fungi compared to plot 7, which was the

one with the lowest frequency (gure 2 and table 2).

Alternaria

44%

Aspergillus

Cladosporium

23 %

Fusarium

Lasiodiplodia

Sterile mycelium

16%

Nigrospora

5 %

Stemphylium

5 %

Trichoderma

Ulocladium

Figure 2. Percentage relative frequency of genera of endophytic

fungi isolated from blueberry plants (Vaccinium

corymbosum L.) from production plots in the province

of Cañete, Peru, 2020.

Table 2. Absolute and relative frequencies with the variables production plots, genera of endophytic fungi, leaves and stems of blueberry

plants.

Production

plots

Genera of endophytic fungi associated with blueberry leaves (H) and stems (T).

Alternaria Aspergillus Cladosporium Fusarium Lasiodiplodia

Sterile

mycellium

Nigrospora Stemphylium Trichoderma Ulocladium Total

H T H T H T H T H T H T H T H T H T H T FA

(%)

Plot 1 3 1 0 0 2 0 0 0 0 0 3 0 0 0 2 0 0 0 0 0 11 8.27

Plot 2 6 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 14.29

Plot 3 1 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 1 0 0 4 11.28

Plot 4 2 0 0 0 0 2 0 0 0 0 1 0 0 0 0 0 1 0 0 0 6 12.03

Plot 5 4 1 0

0 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 8 5.26

Plot 6 0 2 0 0 2 1 0 0 0 0 3 0 0 0 0 1 0 0 0 0 9 7.52

Plot 7 3 0 0 0 3 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 3.01

Plot 8 6 5 0 0 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 14 4.51

Plot 9 4 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 6.02

Plot 10 4 4 0 0 1 2 3 0 0 0 1 0 1 0 2 0 0 0 1 0 19 6.77

Plot 11 4 0 0 0 0 1 0 0 0 1 2 3 1 0 1 0 0 2 0 0 15 5.26

Plot 12 8 0 0 0 1 2 0 1 0 0 2 2 0 0 0 0 0 0 0 0 16 10.53

Plot 13 0 0 0 1 0 3 0 0 0 0 0 2 0 0 0 0 0 1 0 0 7 5.26

Total FA

45 13 0 1 11 19 3 1 0 1 12 9 4 2 5 1 1 4 1 0 133

Total FR

(%)

33.8 9.77 0 0.75 8.27 14.3 2.26 0.75 0 0.75 9.02 6.77 3.01 1.5 3.76 0.75 0.75 3.01 0.75 0

100

FA: Absolute frequency RF: Relative frequency expressed as a percentage.

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Of the total isolated strains and genera identied in blueberry

plants, Aspergillus and Lasiodiplodia were only found in stems;

Similarly, Ulocladium only in leaves compared to the other

genera that were isolated in both organs. This is how, in leaves,

Alternaria was the most frequent genus with 54.88% followed

by Cladosporium with 13.41%, the opposite resulted in stems,

the genus Cladosporium was the most frequent with 37.25%

followed by Alternaria with 25.49%; while the least frequent were

Ulocladium and Trichoderma with 1.22% in leaves, and Aspergillus,

Stemphylium and Lasiodiplodia with 1.96% in stems (gure 3).

Alternaria (54.88)

Alternaria (25.49)

Aspergillus (0.00)

Aspergillus (1.96)

Cladosporium (13.41)

Cladosporium (37.25)

Fusarium 3.66

Fusarium (1.96)

Lasiodiplodia (0.00)

Lasiodiplodia (1.96)

St erile mycelium (14.63)

St erile mycelium (17.65)

Nigrospora (3.92)

Stemphylium (6.10)

Nigrospora (4.88)

Trichoderma (7.84)

Ulocladium (1.22)

Trichoderma (1.22)

Ulocladium (0.00)

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Leaf

Stem

Relative frecuency (%)

Figure 3. Percentage relative frequency of genera of endophytic

fungi at the level of leaves and stems of blueberry

plants (Vaccinium corymbosum L.) in the province of

Cañete, Peru, 2020.

The colonization of endophytic fungi in the tissues of three-year-

old blueberry plants, under a production system in polyethylene

bags, varies from 5.56% to 19.91%, with the Alternaria genus

having the highest colonization, followed by Nigrospora; while the

genera Aspergillus, Lasiodiplodia and Ulocladium presented the

lowest colonization (table 3).

Table 3. Percentage of colonization of genera of endophytic

fungi isolated from stems and leaves of blueberry

plants in the province of Cañete, Peru, 2020.

Genus

Endophytic colonization (%)

Leaves Stems Plant

Alternaria 10.88 9.03 19.91

Aspergillus 0 5.56 5.56

Cladosporium 6.12 7.04 13.16

Fusarium 8.34 5.56 13.9

Lasiodiplodia 0 5.56 5.56

Sterile mycelium 6.02 6.26 12.28

Nigrospora 5.56 11.11 16.67

Stemphylium 6.95 5.56 12.51

Trichoderma 5.56 7.41 12.97

Ulocladium 5.56 0 5.56

Of the identied genera, Alternaria and Cladosporium were

the most frequent endophytic fungi followed by sterile mycelium

(microorganisms did not form reproductive structures); likewise,

the greatest diversity was in blueberry leaves. Similar studies

indicate that these genera were also the most frequent endophytes

in soybeans (Impullitti and Malvick, 2013). On the other hand,

Larrain et al. (2000) reported that the highest number of endophytic

fungal taxa isolated were from the leaves of healthy wheat plants.

Likewise, Cladosporium was one of the genera most frequently

isolated from Glycine max leaves (Fernandes et al., 2015). Instead,

Cladosporium cladosporioides was sporadically reported on healthy

leaves of Caesalpinia echinata Lam. (Lima and Cavalcanti, 2014).

They were also reported on the leaves of Vaccinium dunalianum

var. Urophyllum the greatest diversity of endophytic fungi and the

genus Cladosporium was found in branches (Li et al., 2016). In

contrast, a higher number of endophytes were isolated from stem

tissues than from leaves in soybean and corn varieties (Russo et

al., 2016).

Species of Fusarium, Aspergillus, Alternaria and Nigrospora

were reported as stem and leaf endophytes in medicinal plants with

a colonization frequency of less than 6.66% (Anitha et al., 2013).

Likewise, the endophytic genera Cladosporium, Nigrospora and

Trichoderma were isolated from leaves of Caesalpinia echinata

Lam. less frequently (Lima and Cavalcanti, 2014). Similarly, in

this investigation, these genera appeared less frequently in the

tissues of blueberry plants, as well as Ulocladium, Lasiodiplodia

and Stemphilium, except for Alternaria and Cladosporium, which

were the ones that presented the highest frequency. On the contrary,

Piontelli et al. (2002) reported the genus Ulocladium as the most

dominant endophyte in all steps and plant substrates. Likewise,

the most frequently isolated species in soybean was Fusarium

graminearum

and Aspergillus terreus in corn (Russo et al., 2016).

Likewise, Ulocladium, Fusarium and Aspergillus sp. were identied

as endophytes of palm hearts (Euterpe precatoriaMart.) (Batista et

al., 2018). On the other hand, Aharwal and Kumar (2016) reported

Aspergillus, Alternaria and Fusarium among the isolated genera

of Ricinus communis. Also, the Nigrospora genus was isolated

from leaves of healthy Vinca rosea plants (Metwaly et al., 2014).

Regarding the genus Lasiodiplodia, there are reports indicating that

it is an endophytic fungus of Caesalpinia echinata Lam. (Lima and

Cavalcanti, 2014); Similarly, strains of Lasiodiplodia theobromae

and Nigrospora sphaerica were identied as endophytes of

Theobroma cacao (Vásquez et al., 2018). Therefore, it would be

conrmed that the genera identied in this research are endophytic

fungi.

These microorganisms colonize inter and/or intracellularly the

plant tissue asymptomatically; however, very little is known about

the diversity and function of these fungi in species of economic

importance (Russo et al., 2016). Thus, Rothen (2015) showed that

not all endophytic fungi isolated from G. max behave in this way,

only 26% behave as endophytes in the strict sense, that is, they do

not cause symptoms, and the rest as endophytes in broad sense

(17%), biotrophic pathogens (40%) and necrotrophic pathogens

(17%) that present symptoms. Therefore, future research is required

for the molecular identication at the species level of the isolated

strains of V. corymbosum and conrm their endophytic capacity.

Thus, it would allow the selection of endophytic and pathogenic

strains, since there are species of the genera isolated in this study

that behave as pathogens of plant species, except for the genus

Stemphylium (1.96)

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Rev. Fac. Agron. (LUZ). 2022, 39(1): e223922. January - March. ISSN 2477-9407.6-7 |

Trichoderma, which is used as a biocontrol agent against many

phytopathogenic fungi. (Citrus, 2005). In addition, in V. corymbosum,

species of Lasiodiplodia (Rodríguez-Gálvez et al., 2020), Nigrospora

sphaerica (Wright et al., 2007), Alternaria alternata (Nadziakiewicz

et al., 2018), Cladosporium tenuiussimum were identied as

pathogens. (Lei et al., 2019), Stemphylium sp. (Flores and Medina,

2012) and Fusarium oxysporum (Moya-Elizondo et al., 2019) with

the exception of the Aspergillus, Ulocladium and Trichoderma genera

that do not cause disease in blueberry plants. On the contrary, these

genera have antifungal activity against phytopathogens (XiaoXue

et al., 2018). There are reports indicating that strains of the same

species have a great diversity of responses to their host. Thus, some

pathogens, before showing disease symptoms, are in a latent phase

within the host tissue (Photita et al, 2004), such is the case of L.

theobromae that behaves as a latent pathogen (Mohali et al., 2005) and

as an endophytic antagonist (Vásquez et al., 2018); likewise, certain

endophytes can become pathogens when the host plant is stressed,

due to excessive humidity or nutrient deciency, which induce the

transition from one way of life to another (Fisher and Petrini, 1992).

In addition, in this research it was found that endophytic fungi

colonized less than 20% of blueberry plant tissues, because the

plants are grown in non-sterile conditions, which means that there

is competition for other microorganisms (De Souza et al. ,2008).

Likewise, the presence of species of endophytic fungi in plants can

be affected by many factors, such as the type of culture, type of tissue

sampled, age of the plants, climate and location in which they were

grown (Impullitti and Malvick, 2013; Russo et al., 2016).

The species of the genus Trichoderma are a group of

microorganisms most investigated for the control of fungal plant

diseases, they colonize the root surface or live as endophytes within

the tissues and have the ability to reduce diseases through their

mechanisms of action (mycoparasitism, antibiosis, competition and

induced systemic resistance), promote plant growth and improve

plant productivity (Bailey and Melnick, 2013). Similarly, Aspergillus

versicolor, an endophyte of Vaccinium dunalianum, has inhibitory

effects on phytopathogenic fungi (XiaoXue et al., 2018) and has

insecticidal activity (Senthilkumar et al., 2014). Also, Alternaria

species as a potential biocontrol agent for plant pathogens (Lou et

al., 2013) and against some agricultural pests (Sharma and Sharma,

2014). On the other hand, Cladosporium omanense has recently been

described as a new endophytic fungus that has the ability to suppress

Pythium aphanidermatum (Halo et al., 2021). Therefore, the isolated

strains of V. corymbosum in the Cañete valley could be possible

biological control agents for pests and diseases of blueberries and

other crops.

Conclusions

For the rst time, nine genera of endophytic fungi (Trichoderma,

Aspergillus, Alternaria, Ulocladium, Cladosporium, Fusarium,

Nigrospora, Lasiodiplodia and Stemphylium) and a group of fungi

called sterile mycelium were isolated and identied by taxonomic

classication in leaves and stems of healthy plants of blueberry variety

biloxi from the Cañete valley in Lima, Peru, the leaves being the ones

that obtained the highest frequency of strains. The genera Alternaria

and Cladosporium turned out to be the most frequent and with the

highest colonization in plant tissues, while Aspergillus, Ulocladium

and Lasiodiplodia were the least frequent.

Acknowledgment

Our thanks to Richard Jorge Yactayo Yactayo and José María

Espinosa Astorayme for their help with samples collection for the

research, and to Yasmin Carmen Arestegui Cantoral and Thalia

Esthepany del Pilar Sarmiento Ruiz for their support in isolating

fungi.

Funding source

Financial support from the Universidad Nacional de Cañete- Perú

through the Minor Experimental Research Project No. 019 (Contract

No. 01-2020-UNDC-PIEM).

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