New Disease Reports
New Disease Reports (2019) 40, 2. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.002]
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Diplodia africana causing twig death on Araucaria araucana, a new host and first record for Chile

M. Zapata 1* and M. Schafer 2

*mario.zapata@sag.gob.cl

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Received: 09 Jun 2019; Published: 01 Jul 2019

Keywords: Botryosphaeriaceae, forest pathology, monkey puzzle tree, phylogeny

Araucaria araucana (monkey puzzle tree) is an endemic conifer restricted to the temperate subantarctic forests of South America, with most of the world population concentrated in Chile, covering an area of 253,715 hectares. As a native forest species, A. araucana has been the object of ongoing phytosanitary surveys by the Servicio Agrícola y Ganadero (SAG), the National Plant Protection Organization of Chile. During surveillance activities in 2018, a botryosphaeriaceous fungus was isolated from a sample of partially dead branches from a tree in Conguillio National Park, located in the Andes, Araucanía Region (38°39'00" S, 71°37'6" W). Pycnidia and conidia produced in vitro on pine needles on water agar after 14 days were identified as Diplodia sp., with conidia hyaline after discharge from pycnidia, non-septate, measuring (26-)31-34.5(-36) × (7-)9.5-12(-13.5) µm (Fig. 1).

To identify the species of Diplodia, sequences of the rDNA ITS region, β-tubulin (BT), and translation-elongation factor 1α (TEF1) gene were obtained using the universal primers ITS1/ITS4, EF1-728/EF2 (Carbone & Kohn, 1999; O'Donnell et al., 1998) and TUB2Fd/TUB4Rd (Aveskamp et al., 2009). The sequences were submitted to GenBank with the Accession Nos. MN046380 (ITS), MN046381 (TEF1) and MN046382 (BT). BLAST analysis limited to type material showed 99.5-100% identities with Diplodia africana (MH863094, KF766397 and KF766129). A multi-locus phylogenetic analysis using maximum parsimony confirmed that the specimen clustered with the D. africana clade (bootstrap = 96%) (Fig. 2). An isolate (SAG-102744-18) was deposited in the Chilean Microbial Genetic Resources Collection, INIA Quilamapu, Chillán, Chile, with the accession number RGM 2718.

The pathogenicity of the fungi was tested by inoculating six three-year-old A. araucana seedlings (approximately 40 cm in height) in April 2019. The stems of the seedlings were wounded using a sterile scalpel 5 cm above the collar root and inoculated with a mycelial plug (5 mm diameter) from a seven-day-old culture grown on 2% malt extract agar. Two seedlings used as control were inoculated with sterile agar plugs. The plugs were sealed with paraffin film. Seedlings were kept outdoors with average minimum and maximum temperatures of 5.4 and 21.5°C, and watered as needed. After four weeks, brown phloem lesions ranging from 7-30 mm in length were observed on the inoculated seedlings and the fungus was consistently re-isolated from all lesions. Additionally, all inoculated seedlings showed foliar necrosis affecting between a few leaves to 50% of the foliage. Control seedlings remained healthy (Fig. 3-4). 

The genus Diplodia includes species that are pathogens, endophytes and saprobes of mostly woody hosts. Some of the pathogenic species include D. africana, which was initially found on stone fruit of Prunus spp. in South Africa (Damm et al., 2007) and later reported causing dieback on Phoenicean juniper in Italy (Linaldeddu et al., 2011). To our knowledge, the current identification of D. africana on Araucaria araucana in Chile constitutes a new host-distribution of the fungus.

Figure1+
Figure 1: Conidia of Diplodia africana: A) conidia on conidiophores, B) conidia after discharge from pycnidia, and C) conidia after four weeks. Bar = 10 µm.
Figure 1: Conidia of Diplodia africana: A) conidia on conidiophores, B) conidia after discharge from pycnidia, and C) conidia after four weeks. Bar = 10 µm.
Figure2+
Figure 2: One of four equally parsimonious trees (54 steps, CI = 0.889, HI = 0.111, RI = 0.910) obtained from multi-locus phylogenetic analysis (ITS-TEF1-BT) for selected Diplodia species. Analysed with PAUP v. 4.0b10 (Swofford 2002). DNA sequences aligned using MAFFT v. 7.0 employing E-INS-i strategy. Bootstrap values ≥75 % shown above nodes (1,000 replicates). Tree rooted with Lasiodiplodia theobromae. T = ex-type.
Figure 2: One of four equally parsimonious trees (54 steps, CI = 0.889, HI = 0.111, RI = 0.910) obtained from multi-locus phylogenetic analysis (ITS-TEF1-BT) for selected Diplodia species. Analysed with PAUP v. 4.0b10 (Swofford 2002). DNA sequences aligned using MAFFT v. 7.0 employing E-INS-i strategy. Bootstrap values ≥75 % shown above nodes (1,000 replicates). Tree rooted with Lasiodiplodia theobromae. T = ex-type.
Figure3+
Figure 3: Lesion on stems of Araucaria araucana seedlings four weeks after inoculation with Diplodia africana (left) and control (right). Arrows show foliar necrosis.
Figure 3: Lesion on stems of Araucaria araucana seedlings four weeks after inoculation with Diplodia africana (left) and control (right). Arrows show foliar necrosis.
Figure4+
Figure 4: Colony of Diplodia africana recovered from the inoculated seedlings.
Figure 4: Colony of Diplodia africana recovered from the inoculated seedlings.

Acknowledgements

The author wishes to thank SAG colleagues Diego Rupaillan for sampling, Mariana Llanos and Abraham Aburto for their technical support.

References

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  6. Swofford DL, 2002. PAUP: Phylogenetic Analysis Using Parsimony (and Other Methods). Version 4.0b10. Sunderland, USA: Sinauer Associates.
To cite this report: Zapata M, Schafer M, 2019. Diplodia africana causing twig death on Araucaria araucana, a new host and first record for Chile. New Disease Reports 40, 2. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.002]

©2019 The Authors