Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites (Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province of Santa Fe. In this study, Zoophthora radicans was the most important pathogen and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This study is the first report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina.Centro de Estudios Parasitológicos y de Vectore

López Lastra, Claudia Cristina

Manfrino, Romina Guadalupe

Salto, César E.

Zumoffen, Leticia

Revista Argentina de Microbiología

English

SEDICI - Repositorio de la UNLP

Rev Argent Microbiol. 2014;46(1):49-52
0325-7541/ © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L. Todos los derechos reservados.
www.elsevier.es/ram
R E V I S TA  A R G E N T I N A  D E
MICROB IOLOGÍA  
BRIEF REPORT
Natural occurrence of entomophthoroid fungi of aphid pests 
on Medicago sativa L. in Argentina
Romina G. Manfrinoa,b,*, Leticia Zumoffena,b, César E. Saltoa, and Claudia C. López Lastrac
a Instituto Nacional de Tecnología Agropecuaria (INTA), Área Investigación Agronomía, Protección Vegetal, Rafaela, 
Santa Fe, Argentina
b CONICET, INTA, Rafaela, Santa Fe, Argentina
c Centro de Estudios Parasitológicos y de Vectores (CEPAVE), UNLP-CONICET, La Plata, Buenos Aires, Argentina
Recibido el 28 de febrero de 2013; aceptado el 22 de enero de 2014
* Corresponding author.
E-mail address: manfrino.romina@inta.gob.ar (R.G. Manfrino).
KEYWORDS
Lucerne;
Aphids;
Entomopathogenic 
fungi
Abstract
Four species of entomophthoroid fungi,  Pandora neoaphidis  (Entomophthorales: 
Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), 
Entomophthora planchoniana  (Entomophthorales: Entomophthoraceae) and Neozygites 
fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis 
trifolii, and Acyrthosiphon pisum and unidentif ed species of Acyrthosiphon on lucerne in 
Argentina. Samples were collected from f ve sites (Ceres, Rafaela, Sarmiento, Monte 
Vera and Bernardo de Irigoyen) in the province of Santa Fe.  In this study, Zoophthora 
radicans was the most important pathogen and was recorded mainly on Acyrthosiphon 
sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This 
study is the f rst report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) 
aphids in Argentina.
© 2013 Asociación Argentina de Microbiología. Published by Elsevier España, S.L. All 
rights reserved.
Ocurrencia natural de hongos entomophthorales de áf dos plaga de Medicago sativa 
L. en Argentina
Resumen
Se encontraron cuatro especies de hongos Entomophthorales, Pandora neoaphidis, Zo-
ophthora radicans , Entomophthora planchoniana  (Entomophthorales: Entomophthora-
ceae) y Neozygites fresenii (Neozygitales: Neozygitaceae) infectando a Aphis craccivora, 
Therioaphis trifolii, Acyrthosiphon pisum  y a especies no identi f cadas pertenecientes 
al género Acyrthosiphon en cultivos de alfalfa (Medicago sativa L.), en la Argentina. Los 
muestreos fueron realizados en cinco sitios (Ceres, Rafaela, Sarmiento, Monte V era y 
Bernardo de Irigoyen) de la provincia de Santa Fe.  Zoophthora radicans fue el patógeno 
PALABRAS CLAVE
Alfalfa;
Áf dos;
Hongos 
entomopatógenos
50 R.G. Manfrino et al 
Aphids (Hemiptera: Aphididae) are among the most suc-
cessful families of insects and many represent serious agri-
cultural pests 11. Four of the ten species of aphids in the 
world that infect lucerne 2 are considered serious pests in 
Argentina, these being Aphis craccivora Koch, Acyrthosi-
phon pisum (Harris), Acyrthosiphon kondoi Sinji and Therio-
aphis trifolii (Monell)13. Aphids feed on phloem sap via ex-
tremely thin maxillary stylets that penetrate phloem sieve 
tubes, greatly reducing the possibility of these insects to 
ingest viruses, bacteria or protozoa from plant surfaces. 
Aphids became relevant due to their capacity of transmis-
sion of several viruses like Alfalfa Mosaic V irus (AMV) and 
other potyviruses, which limit the performance and persis-
tence of plants3. Entomophthoralean fungi can cause lethal 
infections of various aphid species and they belong to the 
group of most effective control agents of natural aphid co-
lonies. The only record of Entomophthoroid fungi of aphids 
on lucerne in South America was found in Uruguay1. Limited 
research efforts have been devoted to investigating the 
entomopathogenic fungi as agents of natural mortality of 
aphids in lucerne crops in Argentina. The aim of this paper 
was to identify and to isolate entomophthoroid fungi of 
aphid pests on M. sativa in the Argentina Pampas. The study 
was not intended to provide quantitative data. The taxo-
nomy of entomophthoroid fungi used here is in accordance 
with the new molecular-based classif cation of these fungi, 
including it in a newly described phylum, Entomophtho-
romycota6.
The survey covered the west of Santa Fe province, in the 
Argentine Pampas (situated between 28-40º S and 68-57º W). 
The Argentine Pampa is a vast region of 52 million ha of 
suitable land for agriculture and cattle production. 
Samplings of insects were conducted in f ve sites from April 
2010 to June 2012 (T able 1). Surveys were occasionally 
carried out in Ceres, Sarmiento and in Bernardo de Irigoyen, 
and weekly in Rafaela and Monte V era. Sampled areas did 
not exceed 500 m 2 per site.  No insecticides or fungicides 
were applied to the parts of the f elds where collections were 
made during the course of the study . Fifteen (15) lucerne 
stems (from 30 to 50 cm each) were collected along both 
diagonals of each f eld. Stems sustaining aphids were placed 
in labeled plastic bags and transported to the laboratory as 
described by Zumoffen et al.14. Lucerne stems were checked 
to evaluate the presence of healthy or infected aphids. The 
plants were later discarded. Samples of healthy living 
aphids were collected and transferred into plastic cups with 
lids (150 cm 3) from where subsamples were transferred 
to microcentrifuge tubes (Eppendorf; 1.5 cm 3). These 
subsamples were preserved in 70% ethanol for further 
identif cation to species level, according to Blackman & 
Eastop’s keys2.
Dead aphids with evidence of external fungal growth 
(showing sporulation) were examined under a stereo 
microscope and an optic microscope to evaluate the 
presence of rhizoids, cystidia, and/or spores. Dead aphids 
without external mycosis signs were placed in P etri dishes 
(60 mm diam) containing a f lter paper moistened with a 
few drops of distilled water (humid chambers), which was 
maintained at 20 ºC for 24-72 h to allow the development of 
overt mycoses. Living aphids with apparent infection signs 
were also disposed in humid chambers and maintained 
under the same conditions detailed above until they showed 
an infection development, f nally checking that aphid 
mortality was caused by Entomophthoralean fungi. Fungal 
structures were mounted in lactophenol-aceto-orcein 
(LPAO) (1:1) or stained with 1% aceto-orcein plus glycerine 
for semipermanent mounts. Measurements of fungal 
structures were made to enable speci f c identif cation. 
Fungal species were identif ed according to taxonomic keys 
and monographs of Humber6 and Keller7,8.
In order to obtain pure cultures, infected aphids were 
placed on a moistened piece of sterile f lter paper attached 
with double coated tape to the lid of a sterile 60 mm P etri 
dish, which was then inverted over the bottom of a sterile 
Petri dish containing SEMA (80% Sabouraud dextrose agar + 
1% yeast extract and 20% of a mixture of egg yolk and skim 
milk)4 plus 40.000 units/ml penicillin G (Merck ®, Germany) 
and 80.000 units/ml streptomycin (P arafarm®, Argentina). 
This assembly was left 12 h in the dark at 22 ± 1 ºC. A sterile 
lid replaced the lid with the attached aphids after 12 h. All 
isolates were incubated at 22 ± 1 ºC with a photoperiod of 
16:8 (L: D).
Only one of the species of Entomophthoroid fungi was 
successfully isolated and maintained in pure cultures. 
Zoophthora radicans  isolates were deposited in the 
Mycological Culture Collection at Centro de Estudios 
Parasitológicos y de Vectores (CEP, La Plata, Argentina) and 
at USDA-ARS Collection of Entomopathogenic Fungal 
Cultures (ARSEF, Ithaca, New York) under access numbers 
CEP 362 and ARSEF 11859 CEP , respectively . Herbarium 
materials such as dried infected specimens and microscope 
slides were deposited in the Mycological Culture Collection 
más importante registrado principalmente en Acyrthosiphon sp. Zoophthora radicans fue 
exitosamente aislado y mantenido en cultivos puros. Este estudio documenta por primera 
vez en la Argentina la presencia de hongos Entomophthorales infectando áf dos en alfalfa.
© 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L. Todos 
los derechos reservados.
Table 1 Location of study sites
Town/City District Latitude S Longitude W
Ceres San Cristobal 29º52’55,38’’ 61º56’25,00’’
Rafaela Castellano 31º12’3,67’’ 61º30’25,83’’
Sarmiento Las Colonias 31º3’24,84’’ 61º10’13,29’’
Bernardo 
de Irigoyen
San Jerónimo 32º10’05,65’’ 61º09’19,38’’
Monte Vera La Capital 31º32’58,21’’ 60º41’34,74’’
 Entomophthoroid fungi of aphid pests on Medicago sativa L. 51
at Centro de Estudios Parasitológicos y de Vectores (CEP, La 
Plata, Argentina). 
Three of the species of aphids observed were infected by 
entomophthoroid fungi: A. craccivora, A. pisum, T. trifolii 
and unidentif ed species of the genus Acyrthosiphon. Four 
species of entomophthoralean fungi were indenti f ed in 
these aphids: Pandora neoaphidis  (R emaudière & 
Hennebert) Humber, Zoophthora radicans (Brefeld) Batko, 
Entomophthora planchoniana  Cornu (Entomophthorales: 
Entomophthoraceae), and Neozygites fresenii (Nowakowski) 
Remaudière & Keller (Neozygitales: Neozygitaceae) (Table 2). 
Fungal infections occurred mainly between May and July 
and, to a lesser extent, during April, September and 
December 2010 and February 2012. Previous studies on the 
phenology of entomophthoroid fungi in populations of 
insects other than aphids recorded that fungal infections 
were more common during autumn-winter in Argentina (in 
the Southern hemisphere, from March to September)9.
Entomopthoralean fungal infections were most frequently 
observed in Acyrthosiphon spp. than in the rest of the  
aphid species collected. Entomophthora planchoniana , 
P. neoaphidis and Z. radicans  were identi f ed infecting 
Acyrthosiphon spp. In our study Z. radicans  was the most  
important pathogen recorded from aphid pests on M. sativa 
and it was successfully isolated from A. pisum. On the other 
hand, Alzugaray et al.1 reported P. neoaphidis as the principal 
mortality agent of aphids in lucerne crops in Uruguay. In this 
study P. neoaphidis was secondary to Z. radicans in occurrence 
and the f rst was identif ed among three aphid pest species.  
Entomophthoralean fungi were reported from other  
Leguminoseae plants related to lucerne, as for example  
N. fresenii  that was recorded from A. craccivora  on faba  
Table 2 Entomophthoralean fungi recorded from M. sativa during 2010-2012
Fungal species Host Locality Date of collection
Entomophthora planchoniana Cornu Therioaphis trifolii Rafaela Sep 16, 2010
Acyrthosiphon sp. Monte Vera Sep 24, 2010
Pandora neoaphidis (Remaudière 
& Hennebert) Humber
Acyrthosiphon pisum Monte Vera Apr 21, 2010
Acyrthosiphon sp. Rafaela May 14, 2010
Aphis craccivora Bernardo de Irigoyen May 13, 2011
Sarmiento May 17, 2011
May 19, 2011
May 26, 2011
Jun 13, 2011
Jul 06, 2011
Jul 13, 2011
May 11, 2012
Zoophthora radicans (Brefeld) Batko Acyrthosiphon pisum Monte Vera May 10, 2010
Acyrthosiphon sp. Rafaela Jun 09, 2010
Aphis craccivora Sarmiento Jun 16, 2010
Ceres Jul 06, 2010
Dec 30, 2010
May 13, 2011
May 18, 2011
May 19, 2011
May 20, 2011
May 27, 2011
Jun 13, 2011
Jun 24, 2011
Jul 06, 2011
Jul 13, 2011
May 27, 2012
Jun 10, 2012
Neozygites fresenii (Nowakowski) 
Remaudière & Keller
Aphis craccivora Monte Vera Feb 28, 2012
52 R.G. Manfrino et al 
bean plants12. Entomophthtora species have been recorded 
to infect A. pisum and T. trifolii on legumes in Australia10. 
In the present research T. trifolii  was only infected  
by E. planchoniana . There are previous records of  
E. planchoniana as pathogen of T. trifolii and A. kondoi in 
New Zealand5.
Pandora neoaphidis , Z. radicans, E. planchoniana  and 
N. fresenii were found to infect  A. craccivora, T. trifolii, 
A. pisum and unidentif ed species of Acyrthosiphon ssp. The 
present study is a preliminary record of Entomophthoralean 
fungi causing infections in natural populations of aphids on 
lucerne crops in the Argentina Pampas.
Conf icts of interest
The authors declare that they have no conf icts of interest.
Acknowledgments
We thank Dr. Richard Humber for conf rming the taxonomic 
identif cation of fungal species and for allowing us to depo-
sit fungal cultures for preservation at the USDA-ARS Collec-
tion of Entomopathogenic Fungal Cultures (ARSEF). We also 
thank the National Research Council (CONICET) of Argentina 
for the partial f nancial support of this research.
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Ocurrencia natural de hongos entomophthorales de áfidos plaga de Medicago sativa L. en Argentina

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Ocurrencia natural de hongos entomophthorales de áfidos plaga de Medicago sativa L. en Argentina

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