Revista Mexicana Ciencias Agrícolas   volume 12   number 3   April 01 - May 15, 2021

DOI: https://doi.org/10.29312/remexca.v12i3.2640

Report of Lasiodiplodia theobromae (Pat.) Griffon and Maubl.
in citrus trees in Tamaulipas

Héctor Flores Hernández¹

Juan Flores Gracia¹

Sostenes Edmundo Varela Fuentes²

Amado Pérez Rodríguez3

Ausencio Azuara Domínguez¹

Abraham Monteon-Ojeda3§

1National Technological Institute of Mexico-Technological Institute of Ciudad Victoria-Division of Postgraduate Studies and Research. Boulevard Emilio Portes Gil no. 1301, Ciudad Victoria, Tamaulipas, Mexico. ZC. 87010. (floresgracia@yahoo.com.mx; azuarad@gmail.com). 2Autonomous University of Tamaulipas-Faculty of Engineering and Sciences-Victoria University Center, Ciudad Victoria, Tamaulipas, Mexico. ZC. 87149. (svarela@uat.edu.mx). 3Postgraduate College-Institute of Plant Health. Highway Mexico-Texcoco km 35.5, Montecillo, State of Mexico, Mexico. ZC. 56230. (perez.amado@colpos.mx; abraham.monteon@gmail.com).

§Corresponding author: hfhmex@hotmail.com.

Abstract

This study determined the presence of Lasiodiplodia theobromae in citrus trees with deterioration and descending death in the central area of Tamaulipas, Mexico in 2017 and 2018. In the study area, plant samples were collected in the trees of valencia orange, grapefruit, mandarin and Italian lemon. In the laboratory, the samples were processed and the fungi were isolated and identified with taxonomic keys and by genetic analysis of the transcribed internal spacer and elongation factor 1 alpha TEF1. 33 strains of fungi were isolated in the 19 commercial citrus orchards, 26 belonged to the genus Lasiodiplodia sp., 3 of Botryosphaeria sp., 1 of Colletotrichum sp., 1 of Cyphellophora sp., 1 of Fusarium sp., and 1 of Nigrospora sp. Of the strains of the genus Lasiodiplodia sp., these were identified as L. theobromae in citrus trees with gummosis, rot, descending death in branches and fruit mummification.

Keywords: fungi, genetic analysis, rot.

Reception date: February 2021

Acceptance date: April 2021

Introduction

The fungus Lasiodiplodia theobromae (Pat.) Griffon and Maubl. it is classified within the Ascomycetes, in the order Botryosphaeriales and family Botryosphaeriaceae (Schoch et al., 2006; Slippers et al., 2013). It is a saprophyte and endophyte fungus and is considered a latent pathogen. However, it is pathogenic when the host is weakened or stressed (Rubini et al., 2005; Mohali et al., 2005).

The fungus causes regressive death of the branches, lesions on the stems, generates rubber and post-harvest fruit rots (Sánchez et al., 1989; Herrera et al., 1993). In the field, in the crop of valencia orange and ruby red grapefruits, damage by L. theobromae consists of defoliation and presence of rubber in the secondary branches, necrosis of the phloem and xylem.

On the other hand, it also causes damage to the crop of mamey [Pouteria sapota (Jacq.) H. E. Moore and Stearn], grape (Vitis vinifera L.), avocado (Persea americana Mill), kumquat [Fortunella margarita (Lour.) Swingle] and mango (Mangifera indica Lin.) (Urbez y Gubler, 2011). In these trees, L. theobromae can occur alone or in interaction with Colletotrichum sp., Fomitoporia maxonii and Fusarium sp. Such interaction causes chlorosis, necrosis, screened, cankers, blights, wet or dry rots, mummifications, wounds, scabs and withering (Kimati et al., 1995).

Also, the interaction of L. theobromae with Fomitiporia maxonii Murrill has been demonstrated; Alternaria citri Ellis and Pierce; Colletotrichum gloeosporioides (Penz.) Sacc; Fusarium solanii (Mart.), Appel and Wollen, Fusarium sp., Dothiorella sp., Phytophthora (Oomycetes), Cephaleuros virescens, Kunze) and C. Liberibacter asiaticus (Cabrera et al., 2012, Cabrera et al., 2017). Therefore, in this work the presence of Lasiodiplodia theobromae was determined in citrus trees with deterioration and dieback in the central area of Tamaulipas, Mexico in 2017 and 2018.

Materials and methods

Study area

The research work was carried out in 2017 and 2018 in 19 commercial citrus orchards located in the municipality of Güémez, Llera de canales, Padilla and Victoria Tamaulipas, Mexico (Table 1).

Table 1. Location of the sites where the study was carried out.

Collect plant material

The plant material was collected in 27 Italian lemon trees (Citrus limon Burm), 10 valence orange (Citrus sinensis L. Osbeck), 1 red double grapefruit (Citrus paradisi Macfad) and 1 mandarin (Citrus reticulata Blanco). The trees had the following symptoms: rot of the wood, dried branches, rotten fruits with black mycelium, rot and cankers in the bark of the branch, dried branches with rubbers and leaves with black and white mycelium. In each structure (wood, branch, fruit and leaf), 200 g of material was collected. The samples were poured into polyethylene bags labeled and transported to the laboratory of Molecular Biology and Biotechnology of the postgraduate degree in biology of the Institute of Technology of Victoria, Tamaulipas.

Isolation and taxonomic identification of phytopathogenic fungi

Five 0.5 cm tissue sections were cut from each sample. These were disinfected with sodium hypochlorite at 1% by 3 min, washed with sterile distilled water, dried and sown separately in potato-dextrose-agar (Cabrera et al., 2012). The crops were incubated at 25 °C with white light for 3 days. Of the insulation obtained, monospiric cultures were made in agar water (18 g agar dissolved in one liter of distilled water). Afterwards, fungi were identified at the genus and species level based on taxonomic characteristics published by Punithalingam, (1976); Burgess et al. (2006); Barnett and Hunter (2006).

DNA extraction and PCR development of L. theobromae

DNA was extracted using the technique of Ahrens and Seemüller (1992). In the extracted DNA, the ITS genomic region (ITS1, 5.8 S and ITS2) and the alpha elongation factor gene (EF-1α) were amplified. The ITS1 region was amplified with the initiators ITS1 (5’-TCCGTAGGTGACTCTGCGG-3’) and ITS4 (3’-TCCTCCGCTTATTGATATGC-5’) and ITS4 (3'-TCCTCCGCTTATTGATATGC-5’) and ITS2 with ITS5 initiators (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (3’-TCCTCCGCTTATTGATATGC-5’) (White et al., 1990). While, the EF-1α was amplified with the initiators EF1F (5’-TGTTGCTGTTAAGGATTTGAAGCG-3’) and EF1R (3’-AACAGTTTGACGCA TGTCCCTAAC-5’) (Rehner and Buckley, 2005).

The mixture of PCR to amplify both regions consisted of: ultrapure water (13.22 µl), buffer solution TBE 1X (2.5 µl), MgCl at 2.5 mM (2.08 µl), dNTPs to 0.2 mM (2 µl), initiators at 20 µmol (2 µl of each), DNA polymerase (Biogenica®) to 1U (0.2 µl) and 1 ml of DNA (80 ng).

The parameters for PCR were: 94 °C for 5 min, 35 cycles of 94 °C for 5 min, 60 °C for 1 min, 72 °C for 15 min and a final cycle of 72 °C for 5 min. The amplified regions were purified with a commercial kit (Promega) and the fragments obtained were sent to sequence to the Faculty of Sciences of the National Autonomous University of Mexico (UNAM).

Genetic analysis

The sequences obtained from the ITS region and EF-1α were edited to build consensus, lined up, with the Clustal W algorithm included in The BioEdit v7.0.9 software and compared to the sequences deposited at the National Center for Biotechnological Information (NCBI). The generated matrices were used to perform phylogenetic analysis with the 'nearest neighbor' method based on the Neighbor-Joining method (Saitou and Nei, 1987). Selecting the sequences with the greatest similarity and equality in the size of the fragment in order to calculate the evolutionary distance (Tajima and Nei, 1984) in the MEGA7 software (Kumar et al., 2016).

Results

The presence of the fungus was determined at prospective sites characterizing tree damage and symptoms (Figure 1). 33 strains of fungi were isolated in the 19 commercial orchards (Table 2). Of which, 20 strains were collected in Italian lemon, 11 in valence orange, 1 in mandarin and 1 in grapefruit crop. In the trees, 13 strains were collected in the trunk, 13 in the branches, 3 in fruits, 3 in the root and one in the leaves.

Figure 1. Symptoms of citrus damage: gummosis in branches (a), damage to bark and wood (b) and (c); rot in fruit with mycelium growth (d).

Table 2. Record of symptoms in citrus trees sampled in the citrus zona of Tamaulipas, Mexico.

Of the total strains collected, 26 belonged to the genus Lasiodiplodia sp., 3 of Botryosphaeria sp., 1 of Colletotrichum sp., 1 of Cyphellophora sp., 1 of Fusarium sp., and 1 of Nigrospora sp.

Of the strains of the genus Lasiodiplodia sp., 17 strains were isolated from Italian lemon crop, 7 in valencia orange, 1 in mandarina and 1 in grapefruit. In tree structures, 13 strains were collected on the trunk, 10 on the branches and 3 on the fruits. While the three strains of the genus Botryosphaeria sp. were collected in the branches of the valencia orange crop. In contrast, the strain Colletotrichum sp., Cyphellophora sp. and Fusarium sp., was collected from the root of the Italian lemon trees and the strain of the genus Nigrospora sp., was collected on the leaf of this same citrus species (Table 3).

Table 3. Distribution of isolated fungi in the sampled structures of the tree.

On the other hand, in the case of strains of the genus Lasiodiplodia sp., the colonies in the culture medium at 10 days developed a cottony mycelium white and abundant, after 16 days it changed to a dark gray tone (Figure 2) from there, these presented paraphyses hyalines, pycnidia alone or added in the stromatic tissue, immature hyalin conidia, ellipsoid, granulose and truncated-based. As well, dark brown mature conidia, ellipsoid, with longitudinal stretch marks and truncated base (Figure 3).

Figure 2. Mycelium of Lasiodiplodia sp. (a), at 10 days the color of the mycelium is white and covers the whole box, at 16 days it turned to a gray color with black center (b).

Figure 3. Mycelium (a) and mature conidia (b) and young (c) of Lasiodipodia sp.

Genetic characterization of isolated strains of L. theobromae

Of the isolated strains, 17 consensus sequences were obtained from 542 base pairs from the ITS1/5.8S rDNA/ITS2 region and 17 sequences of 314 base pairs of the elongation factor EF-1α. The seventeen sequences of the region ITS1/5.8S rDNA/ITS2 showed homology of 100% with the species L. theobromae (HM466958). These sequences showed no genetic differences between them. Therefore, they were grouped into a single consensus sequence. This sequence was then recorded at the National Center for Biotechnology Information (NCBI) under access number MK886711.

On the other hand, the 17 sequences of the EF-1α elongation factor showed no genetic differences between them. For this, they were grouped into a single consensus sequence. These were then compared with sequences of L. theobromae downloaded from the NCBI. The sequence show homology of 100% with the species L. theobromae. Finally, the analyzed sequence was deposited in the NCBI with access number MK531139.

The construction of the phylogenetic tree, allowed to group the 17 ITS sequences found in eight groups or clades according to the homology in the sequences (Figure 4). While in the phylogenetic tree of the TEF1 alpha sequences, they were grouped into three main clades (Figure 5). Both constructions show a close genetic relationship between the isolates, although the samples came from different municipalities and tissue type. Evolutionary history was inferred using neighbor-joining method. The tree is drawn at scale, with the length of the branches in the same units as the evolutionary distances used to infer the phylogenetic tree.

Evolutionary distances were calculated using the composite maximum probability method. The analysis involved 17 nucleotide sequences. The codon positions included were 1st + 2nd + 3rd. All positions containing missing data were deleted. There was a total of 507 positions in the final dataset. Evolutionary analyses were performed in Mega 7.

Figure 4. Phylogenetic analysis generated from the sequences of the ribosomal ITS region of the isolated trains of L. theobromae. The dendogram was obtained from the analysis by the ‘nearest neighbor’ method based on the Neighbor-Joining method using the Mega 7 program.

Figure 5. Phylogenetic analysis generated from the EF-1α elongation factor sequences of the fungus L. theobromae. The dendogram was obtained from the analysis by the ‘nearest neighbor’ method based on the Neighbor-Joining method using the Mega 7 program.

Discussion

In different parts of the world the fungus L. theobromae is reported in citrus trees (Al-Sadi et al., 2014; Adesemoye et al., 2014; Rodríguez et al., 2016). In Mexico, L. theobromae has been reported causing different diseases in several mainly fruit crops. In this work, the fungus was isolated from the crop of valencia orange, Italian lemon, mandarin and grapefruit in the municipality of Güémez, Llera de Canales, Padilla and Victoria Tamaulipas.

In the municipality of Llera de Canales, Polanco et al. (2019) reported this fungus in the trees of valencia orange. While, in the present work, the fungus was reported in the Italian lemon crop and valence orange in the commercial orchard ‘The Angelicas’ and ‘The Cecilia’. These authors also reported the fungus in the valencia orange crop in General Terán and Montemorelos, Nuevo León.

In sites where the fungus has been recorded, it has commonly been associated with dieback and has been constantly isolated from the branches, bark, vascular tissue and fruits of affected plants (Mullen et al., 1991; Moghal et al., 1993; Mohali et al., 2005). For example, in Venezuela, L. theobromae was isolated from citrus trees with symptoms of dieback and gummosis (Ferrari et al., 1996). In China, L. theobromae generated gummosis in Jatropha podagrica plants (Fu et al., 2007).

While, in India this fungus was the causal agent of root rot and collar rot disease in J. curcas (Latha et al., 2009). In this work, en the trees of both citrus species, the fungus was collected in branches and trunks with xylem rot, in bark and dried branches with the presence of rubber. While, Polanco et al. (2019) reported in trees with symptoms of dieback and necrosis in the trunk and branches. Cedeño y Palacios (1992), mention that L. theobromae produces gummosis and lesions in citrus plants, symptoms similar to those observed in the field.

Of the total strains collected, 26 belonged to the genus Lasiodiplodia sp., 3 of Botryosphaeria sp., 1 of Colletotrichum sp., 1 of Cyphellophora sp., 1 of Fusarium sp. and 1 of Nigrospora sp. Of the strains of the genus Lasiodiplodia sp., 17 strains were isolated from Italian lemon crop, 7 in valencia orange, 1 in mandarin and 1 in grapefruit. Cabrera et al. (2012) in their study describes that citrus plantations in a state of stress and deterioration are important sources of phytopathogenic fungal inoculums. Tropical fruit trees are hosting a large number of these agents that cause serious damage to the different organs of these plants, reduce their productive life, yield and can cause the death of these plants.

In the case of the dieback of citrus branches, this is not an exclusive disease of this fungus, as the species of L. theobromae are presented in conjunction with N. mangiferum and N. parvum of the family Botryosphaeriaceae and cause tree decline disorders and peduncle rots of mango crop (Sakalidis et al., 2011).

In addition to interaction with other phytopathogenic fungi, L. theobromae interacts with HLB, Cabrera et al. (2017) suggests that C. liberibacter may in some way affect the plant's resistance or immunity mechanisms to certain pathogens such as fungi and algae among others and cause the disease. In this sense, the results infer that the bacteria could cause immunodeficiency in citrus plants. The degree of incidence of dry branches appears to be based on the progression of the disease and the degree of weakening of the plant, mainly those that manifest HLB symptoms.

Positive interaction and accelerated deterioration until death have been demonstrated, which citrus trees suffer when affected at the same time by HLB and L. theobromae. It was found, through inoculation trials of these fungi in healthy and sick plants with HLB, that plants with the fungus and HLB were the most affected and exhibited a more severe dieback (Cabrera et al., 2012). In this study, L. theobromae was collected in trees with the presence of the bacterium C. liberibacter asiaticus in psyllid; this may accelerate the final deterioration of the sick tree.

Nariani and Singh (1971) attributed to fungi C. gloeosporioides; L. theobromae and Fusarium sp., accelerated deterioration and dieback of plants following defoliation caused by HLB. In this sense, these pathogens, most present in citrus fruits, could also be considered to play an important role in initial defoliation. Considering that HLB-affected plants show considerable fruit abscission, with a premature drop of 60% to 70%. This would allow inferring that other fungi, such as those mentioned above and not only the bacterium C. liberibacter asiaticus, could be the main responsible for both the fall of fruits and other symptoms in plants with a complex pathogenic situation (Gottwald et al., 2012; Cabrera et al., 2017).

Conclusions

The results of symptomatologic, morphological, ITS sequence analysis and elongation factor 1α, determined that L. theobromae is present and it is associated with the symptoms of mummification and rot of fruits, gummosis, rot and dieback in branches and trees of different citrus varieties, in addition were detected C. gloesporoides, C. eucalipti and F. keratoplasticum in rotten roots of trees with the presence of L. theobromae where a possible association with the deterioration of the trees is evident. This document is the first report on L. theobromae in the citrícola production center region in Tamaulipas.

Acknowledgments

The authors thank CONACYT for its support in carrying out this research work.

Cited literature

Adesemoye, A. O.; Mayorquin, J. S.; Wang, D. H.; Twizeyima, M.; Lynch, S. C. and Eskalen, A. 2014. Identification of species of Botryosphaeriaceae causing bot gummosis in citrus in California. Plant Dis. 98(1):55-61.

Ahrens, U. and Seemüller, E. 1992. Detection de DNA of plant pathogenic mycoplasmalike organisms by polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology. 82(8):828-832.

Al-Sadi, A. M.; Al-Ghaithi, A. G.; Al-Fahdi, N. and Al-Yahyai, R. 2014. Characterization and pathogenicity of fungal pathogens associated with root diseases of citrus in Oman. Inter. J. Agric. Bioly. 16(2):371-376.

Barnett, L. H. and Hunter, B. B. 2006. Illustrated genera of imperfect fungi. fourth (Ed). Am. Phytopathol. Soc. St. Paul, Minnesota, USA. 218 p.

Burgess, T. I.; Barber, P. A.; Mohali, S.; Pegg, G.; De Beer, W. and Wingfield, M. J. 2006. Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology. Mycologia. 98(3):423-435.

Cabrera, R. I.; Betancourt, M.; Ferrer, J.; Hernández, O.; Reyes, N. A.; Piñero, J.; Pérez, V.; Llauger, R. y Herrera, S. 2017. Recuperación de una plantación de naranjo [Citrus sinensis (L.) Osb.] cv. ‘Valencia Late’ con huanglongbing y otras enfermedades. Rev. Inter. Cítricos. 435(1):71-81.

Cabrera, R. I.; Ferrer; J.; Peña I. y Zamora, V. 2012. Lasiodiplodia theobromae (Pat.) Griffon & Maubl., sintomatología, afectaciones e impacto en la citricultura cubana actual. Rev. Levante Agríc. 51(412):254-261.

Cedeño, L. y Palacios, P. E. 1992. Identificación de Botryodiplodia theobromae como la causante de lesiones y gomosis en cítricos. Fitopatol. Venezolana. 5(1):10-13.

Ferrari, F. D.; Ochoa, C. F. M. and Subero, M. L. J. 1996. Dieback and gummosis induced by Lasiodiplodia theobromae (Pat.) Griffon and Maubl. on three citrus tree species. Anales de Botan. Agricola. 3(1):46-49.

Fu, G.; Huang, J. G.; Wei, G. Q.; Yuan, J. G.; Ren, W. H. and Cen, Z. L. 2007. First record of Jatropha podagrica gummosis caused by Botryodiplodia theobromae in China. Aust. Plant Dis. 2(1):75-76.

Gottwald, T. R.; Graham, J. H.; Irey, M. S.; McCollum, T. G. and Wood, B. W. 2012. Inconsequential effect of nutritional treatment on huanglongbing control, fruit quality, bacterial titer and disease progress. Cron Protection. 36(6):73-82.

Herrera, L.; Grillo, H.; Pulgarón, A.; Ruiz, B. y Santos, G. 1993. La poda de saneamiento en cítricos. Centro Agrícola. 20(1):33-44.

Kimat, H.; Bergamin F. A. y Amorim, L. 1995. Manual de fitopatologia:  princípios e conceitos. Controle químico Editora Agronômica Ceres Ltda. 3ra. Ed.  Vol. 1. São Paulo, SP, Brazil. 761-784 p.

Kumar, S.; Stecher, G. and Tamura, K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33(7):1870-1874.

Latha, P. V.; Prakasam, A.; Kamalakanna, C.; Gopalakrishnan, T.; Raguchander, M.; Paramathma, and Samiyappan, R. 2009. First report of Lasiodiplodia theobromae (Pat.) Griffon and Maubl. causing root and collar rot disease of physic nut (Jatropha curcas L.) in India. Aust. Plant Dis. 4 (2):19-21.

Moghal, S. M.; Shivanathan, P.; Mani, A.; Al-Zidjali, A. D.; Al-Zidjali T. S. and Al-Raeesy, Y. M. 1993. Status of pests and diseases in Oman. Series 1: plant diseases in the Batinah. Muscat: Ministry of agriculture and fisheries. Agricultural Research Center, Rumais First Edition. DGAR, ARC No. 6.

Mohali, S.; Burgess, T. I. and Wingfield, M. J. 2005. Diversity and host association of the tropical tree endophyte Lasiodiplodia theobromae revealed using simple sequence repeat markers. Forest Pathol. 35(6):385-396.

Mullen, J. M.; Gilliam, C. H.; Hagan A. K. and Morgan, J. G. 1991. Canker of dogwood caused by Lasiodiplodia theobromae, a disease influenced by drought stress or cultivar selection. Plant Dis. 75(9):886-889.

Nariani, T. K. and Singh, G. R. 1971. Epidemiological studies on the citrus die- back complex in India. Proc. Indian Natl. Sci. Acad. Ser. 37(5):365-371.

Polanco, L. G.; Alvarado, O. G.; Pérez, O.; González, R. y Olivares, E. 2019. Hongos asociados con la muerte regresiva de los cítricos en Nuevo León y Tamaulipas, México. Rev. Mex. Cienc. Agríc. 10(4):757-764.

Punithalingam, E. 1976. Botryodiplodia theobromae. [Description of pathogenic fungi and bacteria]. Commonwealth Mycological Institute. CAB International CABI Bioscience. No. 52. Bakeham Lane, Egham, Surrey, England. 519 p.  

Rehner, S. A. and Buckley, E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia. 97(1):84-89.

Rodríguez, G. E.; Guerrero, P.; Barradas, C.; Crous, P. W. and Alves, A. 2016. Phylogeny and pathogenicity of Lasiodiplodia species associated with dieback of mango in Peru. Fungal Biology. 121(4):1-14.

Rubini, M. R.; Silva-Ribeiro, R. T.; Pomella, A. W.; Maki, C. S.; Araujo, W. L.; dos Santos, D. R. and Azevedo, J. L. 2005. Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches´ broom disease. Inter. J. Biol. Sci. 1(1):24-33.

Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4(4):406-425.

Sakalidis, M. L.; Ray, J. D.; Lanoiselet, V.; Hardy, G. E. StJ. and Burgess, T. I. 2011. Pathogenic Botryosphaeriaceae associated with Mangifera indica in the Kimberley region of western Australia. Eur. J. Plant Pathol. 130(7):379-391.

Sánchez, N.; Zamora, V.; Castellanos, A. y Casín, J. C. 1989. Estudio de hongos encontrados en ramas dañadas por Elaphidion cayamae (Coleoptera: Cerambycidae). Aislamiento y comportamiento en cinco medios de cultivo. Ciencia y Técnica en la Agricultura, Cítricos y otros Frutales. 12(1):131-139.

Schoch, C. L.; Shoemaker, R. A.; Seifert, K. A. Hambleton, S.; Spatafora, J. W. and Crous, P. W. 2006. A multigene phylogeny of the Dothideomycetes using four nuclear loci. Mycologia. 98(6):1041-1052.

Slippers, B.; Boissin, E.; Phillips, A. J. L.; Groenewald, J. Z.; Lombard, L.; Wingfield, M. J.; Postma, A.; Burgess, T. and Crous, P. W. 2013. Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework. Studies in Mycology. 76(1):31-49.

Tajima, F. and Nei, M. 1984. Estimation of evolutionary distance between nucleotide sequences. Mol. Biol. Evol. 1(1):269-285.

Úrbez, J. R. and Gubler, W. D. 2011. Susceptibility of grapevine pruning wounds to infection by Lasiodiplodia theobromae and Neofusicoccum parvum. Plant Pathol. 60(2):261-270.