Additional species of Aspergillus causing bole rot disease in Agave sisalana

SHORT COMMUNICATION

Additional species of Aspergillus causing bole rot disease in Agave sisalana

Patrícia Oliveira dos Santos¹; Augusto César Moura da Silva²; Élida Barbosa Corrêa³; Valter Cruz Magalhães; Jorge Teodoro de Souza

Universidade Federal do Recôncavo da Bahia, CCAAB, Rua Rui Barbosa 710, Centro, 44380-000, Cruz das Almas, BA, Brazil

^{Author for correspondence}

ABSTRACT

The production of sisal in Bahia, Brazil, has been declining due to the occurrence of a disease know as bole rot. Aspergillus niger was regarded as the only causal agent. In this study A. brasiliensis and A. tubingensis, in addition to Aspergillus niger, identified on the basis of morphological and molecular analyses, were shown to cause bole rot of sisal. Their pathogenicity was confirmed but their significance for the epidemiology of the disease in the field remains unclear.

Key words: β-tubulin, pathogenicity, phylogenetic analysis.

Sisal (Agave sisalana) is originated in the Yucatan peninsula, Mexico and was brought to Brazil in the 1900's for fibre production. This crop was introduced into the semi-arid region of the Brazilian Northeast, where it is grown in approximately 263,472 ha (IBGE, 2012). Bahia State is the main producer, contributing with 94% of the national production, generating 94,000,000 USD per year of revenue. Sisal is a crop that depends mainly on familiar labour and employs 700,000 workers in one of the poorest regions of the country (Silva et al., 2008). Since the last decade, sisal production has been declining due to a disease known as bole rot. Infected plants exhibit wilting and yellowing of leaves and reddening of the stem or bole and base of the leaves followed by death (Sá, 2009). The fungus Aspergillusniger was identified by morphological features as the agent of the disease (Coutinho et al., 2006). Several species of Aspergillus are morphologically indistinguishable, especially the ones in section Nigri (the black Aspergilli) and the Aspergillus niger complex (Giraud et al., 2007). The following species belong to the A. niger complex: A. niger, A. tubingensis, A. luchuensis, A. brasiliensis, A. costaricaensis, A. lacticoffeatus, A. piperis, A. vadensis, A. eucalypticola, A. welwitschiae, and A. neoniger (Perrone, 2007; Varga et al., 2011; Hong et al., 2013). The objective of this study was to isolate, identify and test the pathogenicity of Aspergilli isolated from sisal plants and soil.

Aspergillus isolates were obtained from soil and from diseased plants in the sisal-growing region of Bahia. Soil dilutions or surface-sterilized fragments from sisal stems were plated on potato dextrose agar (PDA) with 6% NaCl, that is semi-selective to Aspergillus (Berjak, 1984). The DNA of the 23 isolates used in this study was isolated by the protocol described by Doyle & Doyle (1990). Random Amplified Polymorphic DNA (RAPD) analysis was done with primers A1, A6 and OPA4 according to Abed (2008). The program FreeTree (Hampl et al., 2001) was used to construct the dendrogram using the distances of Jaccard and the UPGMA method. Seven isolates of different groups were selected from the RAPD analysis for molecular identification by sequencing a fragment of 520 pb of the β-tubulin gene. Primers Bt2a and Bt2b (Glass & Donaldson, 1995) were used for amplification and sequencing, which were done according to standard protocols using an ABI 310 sequencer. Sequences were deposited in public databases and the accession numbers are given in Figure 1. Alignment of the obtained sequences and reference sequences from public databases was performed using the program MAFFT version 6.0 (Katoh et al., 2002). The phylogenetic tree was constructed with the maximum likelihood method implemented in the program MEGA version 5.0, with the Kimura-2 parameter nucleotide substitution model and bootstrap analysis with 1000 resamplings. Pathogenicity tests were done by inoculating each of the seven isolates shown underlined in the phylogenetic tree (Figure 1B) in five 4-month old sisal plantlets grown in polyethylene bags filled with 1 kg of soil under greenhouse conditions. Spore suspensions were prepared by growing the isolates on PDA for 10 days at 25ºC and harvesting the spores with a Drigalsky rod. The concentration of the suspensions was adjusted to 10⁷ conidia/ml in a haemocytometer. The stem of the plantlets was wounded with two 1-mm diameter needles fixed to a wooden base. The needles were 2 cm apart and produced 2 cm deep wounds. Each plantlet was inoculated with 100 µl of each spore suspension. Controls were treated with sterile distilled water. The experiment was installed in a randomized design with five replicates per treatment. Thirty days later, plantlets were removed from the bags and split open to observe the incidence of the disease. This whole experiment was repeated at least three times for each isolate. When symptoms were present, fragments of the diseased plantlet were surface-sterilized and plated on PDA to confirm the identity of the isolates. Furthermore, DNA of the isolates recovered was re-sequenced to compare with the sequences obtained for the isolates originally inoculated.

 

From the 23 isolates obtained in association with sisal plants, one was from a diseased stem, eight from leaves, 10 from soil, and four from the air. RAPD analysis showed that the isolates were grouped into 16 different genetic groups with 100% similarity (Figure 1A). From these 16 groups, only Aspergillus alabamensis (ANS 181) and A. aculeatus (ANS 113) could be distinguished from the others in terms of morphology. Sequence analysis showed the following species in association with sisal: A. niger, A. aculeatus, A. alabamensis, A. tubingensis and A. brasiliensis (Figure 1B). The identity of the sequences varied from 99 to 100% when they were compared with other sequences from the databases. Pathogenicity tests showed that only A. niger, A. tubingensis and A. brasiliensis were pathogenic to sisal (Figure 2). The incidence of the disease was 100% for A. niger, 60% for A. tubingensis and 80% for A. brasilensis. The re-isolations and sequencing of the Aspergilli from lesions of diseased plants confirmed the pathogenicity of A. niger, A. tubingensis and A. brasiliensis as agents of sisal bole rot disease.

Little scientific information is available on sisal in general and specifically on the diseases that affect this plant. Only two earlier reports, Coutinho et al. (2006) and Soares et al. (2006) show that A. niger is the agent of sisal bole rot disease. We show here for the first time that besides A. niger, A. tubingensis and A. brasiliensis are able to cause the disease in sisal. Although species of Aspergillus are known as saprophytes or opportunistic pathogens in peanuts, onions and Welwitschiamirabilis (Moraes et al., 1997; Nunes et al., 1997; Hong et al., 2013), they cause this devastating disease in sisal, with incidence in the field varying from 5% to 65% (Coutinho et al., 2006; Abreu et al., 2007). The species A.tubingensis and A. brasiliensis were isolated from soil and the real significance of these isolates in the epidemiology of the disease remains to be determined. A. alabamensis was found in great numbers in soil and was not able to cause disease in sisal. Our observations indicate an inverse relationship in densities of A. niger and A.alabamensis, suggesting that the latter could act as a natural antagonist. Unfortunately, there is one study showing that A.alabamensis may colonize the lungs of patients with cystic fibrosis (Balajee et al., 2009). This report adds two new potential agents of sisal bole rot disease in the sisal-growing region of Bahia state, Brazil.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support of Fundação de Amparo à Pesquisa do Estado da Bahia - FAPESB, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, and Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq.

Submitted: 19 October 2013

Revisions requested: 5 December 2013

Accepted: 6 February 2014

TPP-2013-0174

Section Editor: Meike Piepenbring

Present address:

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1

Instituto Federal Baiano, Campus Santa Inês, 45320-000, Santa Inês, BA, Brazil

2

Embrapa Mandioca e Fruticultura, 44380-000, Cruz das Almas, BA, Brazil

3

Universidade Estadual da Paraíba, Campus II, 58429-500, Areia, PB, Brazil.

Publication Dates

• Publication in this collection
  05 Aug 2014
• Date of issue
  Aug 2014

History

• Accepted
  06 Feb 2014
• Received
  19 Oct 2013
• Reviewed
  05 Dec 2013