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Molecular and Pathogenic Study of Guignardia Spp. Isolates Associated to Different Hosts

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Molecular and Pathogenic Study of Guignardia Spp. Isolates Associated to Different Hosts Advances in Microbiology, 2014, 4, 116-125 Published Online January 2014 (http://www.scirp.org/journal/aim) http://dx.doi.org/10.4236/aim.2014.42016 Molecular and Pathogenic Study of Guignardia spp. Isolates Associated to Different Hosts Ester Wickert1*, Andressa de Souza2, Rodrigo Matheus Pereira3, Luciano Takeshi Kishi4, Eliana Gertrudes de Macedo Lemos2, Antonio de Goes5 1Estação Experimental de Itajaí, EPAGRI, Itajaí, Estado de Santa Catarina, Brazil 2Departamento de Tecnologia, FCAV/UNESP, Jaboticabal, Estado de São Paulo, Brazil 3UFGD, Faculdade de Ciências Biológicas e Ambientais, Dourados, Estado de Mato Grosso do Sul, Brazil 4Departamento de Genética e Evolução, UFSCAR, São Carlos, Estado de São Paulo, Brazil 5Departamento de Fitossanidade, FCAV/UNESP, Jaboticabal, Estado de São Paulo, Brazil Email: *[email protected] Received November 6, 2013; revised December 6, 2013; accepted December 13, 2013 Copyright © 2014 Ester Wickert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor- dance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual property Ester Wickert et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian. ABSTRACT Fungi of Guignardia genus are commonly isolated from different plant species and most of the time, they are characterized as endophytes. However, some species of this genus, like G. citricarpa and G. psidii are known as causal agents of serious diseases that affect important crops such as Citrus Black Spot and guava fruit rot, re- spectively. They are also responsible for diseases that cause foliar spots in different fruit species and also in other crops, but cause minor damages. Despite evidences that G. mangiferae colonizes different plant species, there are few studies about its genetic diversity associated with different hosts. This work has the objective to characterize Guignardia isolates obtained from different hosts and tissues by RAPD, fAFLP and DNA sequence of ITS1-5.8S-ITS2 region, as well as to develop pathogenicity tests through cross inoculation in citrus and guava fruits. It was observed that molecular markers were able to discriminate isolates of different Guignardia species. Pathogenicity tests showed that G. citricarpa caused CBS symptoms on citrus fruits, but it did not produce any symptoms in guava fruits. G. mangiferae isolates were able to cause rot symptoms on guava fruits, but they have not produced any symptoms on citrus fruits. Guignardia isolates obtained from mango leaves that have not been classified in species have not presented any symptoms in citrus and guava fruits. Although G. mangiferae is com- monly isolated asymptomatically in different plants, this work supports the evidence that this species has a latent pathogen behavior, at least for guava plants. KEYWORDS Fungi; Endophytes; Molecular Markers; Plant Disease 1. Introduction Black Spot (CBS) G. citricarpa [2], and G. psidii species causing fruit rot in guava. The G. psidii species is consi- The Guignardia Genus (Kingdom Fungi, Phylum Asco- dered responsible for fruit rot disease in different plants, mycota, Class Dothideomycetes, Order Botryosphaeria- mainly in postharvest conditions. This fungus species is les, Family Botryosphaeriaceae) encompasses around 330 responsible for great losses in guava fruits in Brazil [3]. known species, many of them with unknown anamorphic However, studies using molecular techniques suggested phase [1]. Many species considered plant endophytic fun- that fungi isolates identified as G. psidii could be in fact gi are classified in this family and genus, among them, G. mangiferae, or also could be conspecific to this cos- the G. mangiferae species, the causal agents of Citrus mopolitan species [4]. It is very common that organisms *Corresponding author. belonging to the same species, when obtained from dif- OPEN ACCESS AiM E. WICKERT ET AL. 117 ferent hosts tend to be identified according to the host, re- logia from the Faculdade de Ciências Agrárias e Vete- sulting in taxonomy mistakes and redundancies in data- rinárias/UNESP, Campus de Jaboticabal/São Paulo State, bases. Brazil. Despite causing foliar spots in mango (Mangifera in- dica), G. mangiferae was isolated in a wide range of dif- 2.2. Molecular Studies ferent hosts, and it was considered endophytic because of DNA of the isolates was obtained according [13] proto- the symptomless tissues from which it was isolated. These col. Molecular markers were obtained according the con- hosts include Brazilian tropical plants like Apidosperma ditions described below. polineuron, Anacardium giganteum, Myracrodreun urun- • RAPD markers. RAPD-PCR reaction was carried out deuva, Spondias mombin, Bowdichia nítida and Cassia using 30 ng of genomic DNA, PCR Buffer 1X (50 occidentalis [5]. Citrus plants are also known as hosts of mM KCl, 200 mM Tris-HCl, pH 8,4) (Invitrogen, CA, G. mangiferae [4,6,7], and it is considered endophytic to USA), 2,5 mM MgCl (Invitrogen, CA, USA), 200 this plant species because none symptom is related to this 2 µM dNTP (Invitrogen, CA, USA), 1,5 U Taq DNA fungus in this host. Isolates obtained by these authors polimerase (Invitrogen, CA, USA), 10 pmol of each were identified by DNA sequence of ITS rDNA (ITS1- primer and sterile water to complete the final volume 5.8S-ITS2). Other G. mangiferae plant hosts, like Suri- of 20 µL. Amplification was done on PTC 100 Pro- nam cherry (Eugenia uniflora) and Brazilian grape tree gramable Thermal Controler (MJ Research, Inc.,) ter- (Myrciaria cauliflora) [4] were also identified in Brazil. mocycler with a initial denaturation step of 92˚C dur- Other known G. mangiferae hosts are mango (Mangifera indica L.), banano (Musa sp.) [8], eucaliptus (Eucalytus ing 3 min., 47 cycles of (92˚C during 1 min., 36˚C sp.) [4], and different Ericaeae plants [9]. during 1 min. e 45 s e 72˚C during 2 min.) and a final The use of molecular tools to identify and to charac- cycle of 72˚C during 7 min. Primer kit of Operon Te- terize fungal isolates arouses interest, mainly because of chnologies Inc. was tested to search for primers with its quickness when compared to conventional techniques. good amplifications and polymorphism. Electropho- RAPD (Random Amplified Fragment Lenght Polymor- resis was done on 1.2 % agarose gel using TEB 1X phisms) are the mostly used classes of markers [10] be- buffer (89 mM Tris; 89 mM Boric acid; 2.5 mM cause they are a good method to identify genetic diversi- EDTA, pH 8.3), with ethidium bromide (0.5 µg/ml) ty among different organisms in a short period of time during 1 h 30 min. under 90 Volts. As ladders, 1 kb and low costs. The fAFLP (fluorescent Amplified Frag- DNA ladder plus (Invitrogen, CA, USA) and 100 pb ment Lenght Polymorphism) markers possess advantages DNA ladder (Invitrogen, CA, USA) were used and over other techniques as a tool to identify high levels of gel images were visualized with Gel-Doc 1000 (Bio- genetic diversity and also because it allows reproducibil- Rad, CA, USA) equipment. Data was scored in a bi- ity, fastness and reliability [11]. nary matrix 0 representing absence of bands and 1 the Studies about phylogeny and molecular system of fun- presence of a band for each position. Binary matrix gi have been done using ITS rDNA, because of the high- was analized by PAUP (Phylogenetic Analysis Using er number of random copies of this sequence dispersed in Parcimony-versão 3.01) [14] software to convert it in the genome and the uniformity of them, which is gener- a distance matrix that was used to build the similarity ally maintained by harmonic evolution [12]. The use of phylogram by MEGA (version 4.0) [15] software us- this region proved to be efficient for classifying fungi of ing the Distance Method with Neighbour Joining [16] Guignardia genus in species and also to infer the genetic groupment algorithm. diversity among isolates [4]. • fAFLP markers. These markers were obtained using This work has the objective to characterize Guignardia the “AFLP Microbial Fingerprinting Kit” (Applied Bi- isolates from different hosts by rDNA ITS1-5.8S-ITS2, osystems do Brasil Ltda.), according manufacturer RAPD and fAFLP molecular markers, as well as to carry instructions. fAFLP-PCR products were added with out pathogenic characterization tests by crossed inocula- formamide-loading dye (1.5 ml final volume) and tion in citrus and guava fruits with the same isolates. loaded onto an ABI Prism 377 DNA Sequencer (Ap- plied Biosystems) along with an internal lane stan- 2. Material and Methods dard, GS-500 Rox on ABI Prism 3700 DNA Sequenc- er (Applied Biossytems, Foster City, USA). Frag- 2.1. Isolates Sampling ments were detected and compiled by the ABI Prism Guignardia isolates for this study were obtained from Data CollectionTM (Applied Biosystems) software. different hosts and tissues shown in Table 1. This iso- Gel image files were generated and all the lanes were lates were searched in order to represent the entire col- extracted for making individual electropherograms lection of Guignardia sp. of the Laboratório de Fitopato- using GeneScan (ABI Prism version 1.0) and Geno- OPEN ACCESS AiM 118 E. WICKERT ET AL. Table 1. Isolates characterization by molecular rDNA sequence, OA medium test and crossed innoculation pathogenicity test conducted in Field with citrus and guava fruits. ITS1-5.8S-ITS2 Symptoms Symptoms Isolate GenBank Number Host/tissue OA medium test identification on citrus on guava 35 KF306259 Musa sp./asymptomathic leaf G. mangiferae Without halo No Yes Eu-2 FJ769623 Eucaliptus sp./asymptomathic leaf G. mangiferae Without halo No Yes Lc-29 FJ769698 C. latifolia/asymptomathic leaf G. mangiferae Without halo No Yes G21 FJ769593 P.
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