SciELO - Scientific Electronic Library Online

vol.14 número3Response of bacterial community to simulated nitrogen deposition in soils and a unique relationship between plant species and soil bacteria in the Songnen grassland in Northeastern ChinaSpatial variability of the hydraulic properties of a drip irrigated andisol under blueberries índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados




Links relacionados

  • Em processo de indexaçãoCitado por Google
  • Não possue artigos similaresSimilares em SciELO
  • Em processo de indexaçãoSimilares em Google


Journal of soil science and plant nutrition

versão On-line ISSN 0718-9516

J. Soil Sci. Plant Nutr. vol.14 no.3 Temuco set. 2014  Epub 02-Ago-2014 


Detection of Neofusicoccum nonquaesitum causing dieback and canker in highbush blueberry from Southern Chile


S. Pérez F. 1,2, C. Meriño-Gergichevich3, J. Guerrero C.1*

1Laboratorio de Fitopatología, Facultad de Ciencias Agropecuarias y Forestales. Universidad de La Frontera, Temuco, Chile.

*Corresponding author:

2Dottorando in Scienze e tecnologie Agrarie, ambientali e alimentari. Alma Mater Studiorum - Università di Bologna, Italia.

3Center of Plant, Soil Interaction and Natural Resources Biotechnology. Scientific and Technological Bioresource Nucleus (BIOREN-UFRO), Universidad de La Frontera, Temuco, Chile.


Due to increased incidence of wood fungi from genus Neofusicoccum in highbush blueberry (Vaccinium corymbosum L.) established in various locations in Southern Chile. The objective of this study was the identification of Neofusicocum species in two highbush blueberry cultivars from commercial orchards in Southern Chile. During 2011-12 season, stems with basal cankers and twigs with dieback were collected from cultivars Brigitta and Elliott grown in Panguipulli (39°30'S; 72° 19'W) and Teodoro Schmidt (38°58'S; 73°02'W). Tissues were kept in humid conditions and from cirrus conidia were taken and incubated in PDA medium. The mycelia had a cottony consistency, with colour ranging from greyish-white to black; sub-epidermal pycnidia were eruptive, ostiolate, and brown to black in colour. The unicellular conidia were hyaline, smooth-bordered, coenocytic and septate (1-3), with dense granular content, fusiform and elipsoidal with truncated point, measuring 27.2 — 29.4 (±3.0) μm x 7.7 — 8.4 (±0.9) μm; length/width ratio (L/W) = 3.6 ±0.6 (n=100). The morphometric characteristics corresponded to those of Neofusicoccum nonquaesitum and corroborated genetically (100% homologation) by rDNA sequencing ITS and was recorded in CABI under number IMI-500168. Whereas, the sequencing was deposited into Genbank (accession number JX217819.1). The pathogenicity of the fungus was consistent in twigs and stems of highbush blueberry cultivars Brigitta and Elliott.

Keywords: Highbush blueberry, Neofusicoccum nonquaesitum, wood fungi.

1. Introduction

Highbush blueberry is a fruit-bearing species of economic significance for Chile, with increasing volumes exported both fresh fruit and individually quick frozen (IQF) to North America and some countries in Europe and Asia (Prodorutti et al, 2007; Larach et al., 2009; ODEPA, 2014). Commercial orchards are distributed from the Coquimbo to Los Lagos regions. According to The Government Office in Studies and Agricultural Policies (ODEPA), by 2012 the planted area (hectares) with highbush blueberry per region was: Maule (4,365), Bío Bio (4,280), La Araucanía (1,561), Los Rios (1,519) and Los Lagos (1,141). Total fresh blueberries fruit exported in the 2010-11 season reached 55,012 tons (ODEPA, 2014).

On the other hand, plant health is a crucial factor to obtain a better yield, quality and phytosanitary conditions to blueberry fruit exportation. In recent years, it has been observed an increase of incidence in twigs dieback and stem cankers, associated to wood fungi in highbush blueberry plantations, particularly in the southern Chile; Espinoza et al. (2009) reported between 15 and 45% in wood fungi incidence on blueberry orchards. Currently, this situation is critical due to the severe attack may occur on various cultivars, and because wood fungus control strategies are not fully developed for highbush blueberries. These dieback and cankers have been asso-ciated to a complex of fungal species of genera Botryosphaeria, Neofusicoccum, Pestalotiopsis, Diaporthe and Phoma (Pérez et al, 2010; Mc Donald and Eskalen, 2011).

The taxonomy of the genus Botryosphaeria and its anamorphs has been unclear in the past; the taxonomic changes and the permanent influence of the literature regarding these fungi have caused the confusion (Shenoy et al., 2007; Slippers et al., 2007). There are more than 18 anamorph genera associated to Botryosphaeria, most of which have been reduced to synonyms of Diplodia (pigmented and ovoid conidia, with thick walls) or Fusicoccum (hyaline and fusiform conidia, with thin walls) (Perez et al., 2008; Phillips, 2010). Nevertheless, conidia are also continuously detected with intermediate morphometric characteristics, which is why the genus Neofusicoccum was proposed, by DNA sequencing, distinct from Fusicoccum sensu stricto (F. aesculi) and which produces two different types of conidia; as a result, many species in Fusicoccum sensu lato were situated in the genus Neofusicoccum, based on morphometric characteristics but no by genetic analysis (Alves et al., 2005; Crous et al, 2006).

The genus Neofusicoccum, occurs principally in the Southern Hemisphere in angiosperm and occasionally on gymnosperm (De Wet et al., 2008), affecting a broad range of native and cultivated trees and shrubs world-wide. In Chile the following species have been reported for genus Neofusicoccum i.e. N. mediterraneum, N. australe, N. corticosae, N. arbuti and N. parvum (Espinoza et al., 2009; Acuña, 2010; Pérez, 2010). Particularly, N. nonquaesitum has been also reported in Sequoiadendron giganteum in California, USA (Rooney-Latham et al, 2012), Umbel-lularia californica and Prunus dulcis in California (USA) (Mycobank, 2010) and, in this same study, its detection in V.corymbosum was reported in branches of cvs. Brigitta (Nancagua; Región de O'Higgins, Chile) and Elliott (Rio Negro, Región de Los Lagos, Chile) (Inderbitzin et al, 2010).

Also, the species reported include Fusicoccum putrefaciens Shear (Guerrero, 1988; Guerrero, 2001), F. aesculi Corda, ananamorph of Botryosphaeria dothidea (Moug.) Ces. & De Not., B. ribis Grossenbacher & Duggar (Guerrero, 1993; Acuña, 2010) and its anamorph Fusicoccum sp.; and the more recently reported B. australis Slippers, Crous & M.J. Wingf. (Espinoza et al., 2009), B. parva Pennycook & Samuels (Espinoza et al., 2009); Pérez et al., 2010), Neofusicoccum mediterraneum Crous & M.J. Wingf. & A.J.L. Phillips (Espinoza et al., 2009), N. corticosae Crous & Sumerrel (Espinoza etal., 2009), N. arbuti (D.F. Farr & M. Elliott) Crous, Slippers & A.J.L. Phillips (Espinoza et al, 2009), N. australe Slippers, Crous & M.J. Wingf. (Espinoza et al., 2009), N. parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Espinoza, 2008; Pérez et al., 2010) and N. nonquaesitum Inderb., Trouillas, Bostock & Michalaides (Inderbitzin et al., 2010). Thus, the aim of this study was the identification of the Neofusicoccum species associated to dieback and cankers in two highbush blueberry cultivars from commercial plantations in La Araucanía and Los Ríos regions.

2. Material and Methods

2.1. Geographical location, collection of samples and isolation.

During 2011-2012 season, 50 samples of twigs (growing season) and stems (one or two years-old) were cut from the base of bushes (cultivars Brigitta and Elliott) grown in commercial orchards located in Región de La Araucania (Teodoro Schmidt, 38°58'S; 73°02'W) and Región de Los Rios (Panguipulli, 39°30'S; 72°19'W) and processed in the Phytopathology Laboratory, Universidad de La Frontera, Temuco, Chile. All samples collected were superficially disinfected with 2% (v/v) sodium hypochlorite, after were washed twice with sterile distilled water and then kept in a humid chamber at 25°C (±1°C) and 90% R.H. per seven days. The conidia obtained from the pycnidia cirrus were transferred to Petri plates with potato dextrose agar (PDA, Difco) and streptomycin sulphate (300 ppm) incubated in darkness at 25°C (± 1°C). The obtained isolates were purified taking hypha tips in disposed in quadruplicate for each Petri plates.

2.2. Characterization and identification of the isolates

Morphological identification of isolates was made on the basis of the pycnidia and conidia characteristics (size, shape, color, partitioning, wall thickness and conidia texture) by references from the descriptions of Slippers et al. (2004a,b) and Oliveira et al. (2010); and afterwards corroborated genetically by internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2-28S rRNA) sequence data compared with sequences Genbank by the Centre for Agricultural Bioscience International (CABI), United Kingdom.

2.3. Pathogenicity test

Pathogenicity test was carried out with the isolated CABI IMI-500168, on healthy plants (two years-old) grown in greenhouse conditions (21°C ±2°C, 80% R. H., and 24h of light). Three twigs and stems by plant to each one Brigitta and Elliott, were superficially disinfected with 70% ethanol (v/v) per 30s, and washed once with sterile distilled water. Around to vegetative buds with sterile scalpel was created a wound and inserted by plugs cut from actively growing mycelia on PDA medium. The lesions were covered and sealed with moistened cotton-wool and waterproof tape (Parafilm) during six days.The longitude of the lesion was measured until 21 days and reisolations of fungus were obtained from the adjacent tissue to lesion. Control treatment with PDA plug was maintained under same conditions. Previous studies from our research group, they were carried out on the development of these fungi in different plant substrates (pine needles, apple and kiwi fruits), verifying information that have noted remarkable variability in the phenotypic and morphometric characteristics of this fungus.

3. Results and Discussion

3.1. Detection of Neofusicoccum in commercial plantations

In commercial orchards, diseased plants were characterized by yellowing and fading of foliage, dieback, unilateral stem death, and basal cankers preferably, vascular discoloration and plant death (Figure 1a); the erupting pycnidia were developed on Stem canker (Figure 1b). Mostly cankers with symptoms of fungus were detected at the base of semi and lignified stems. The severity of the necrotic lesions that occurred in young tissues, and a lesser extent in lignified highbush blueberry tissues, may be attributed to a greater translocation of water and nutrients, presence of turgid cells, and less plasmatic membrane resistance to mechanical action exerted by specialized structures of fungi (Ahimera et al., 2004; Úrbez-Torres et al., 2013). According to Agrios (2005), the necrosis may be attributed to the production of phytotoxins, which are extremely toxic even in low concentrations. Proffer (1989) and Rayachhetry et al. (1996) consigned that Neofusicoccum affects cell membrane permeability and enzymes can be detected in connection with lesions produced on one side of stem. This might be explained by fungal invasion in vascular tissues: the mycelium moves under epidermis of the stem, in many cases only on one side, since the xylematic vessels are blocked by hyphae and tyloses (Biggs and Britton, 1988).

Figure 1. a) Basal cankers in stem (left), b) black erupting pycnidia (10X), c) coenocytic and septate conidia (1-3) (40X), d) olive gray mycelium with cottony growth.

3.2. Morphometric characterization

Pycnidia obtained from pine needle after 21 days, were eruptive, colored brown to black, with diameter ranging from 320 to 480 μm (n=20), agglomerations and hyaline conidiophores, without ramifications. Baskarathevan et al. (2009) noted that N. parvum and N. luteum, subsequent to the inoculation with mycelium, did not produce pycnidia on pine needle substrate, which is consistent with the results of this study. Conidia were unicellular, hyaline to brownish, dense granular content, coenocytic , fusiform and elipsoidal, ovoid base and slightly truncated point (Figure 1c). The reference measurements were in [(22.0-) 27.2-29.4 ±3.0 (-34.0) μm x (6.0) 7.78.4 ±0.9 (-10.0)] μm; (L/W) = 3.6 ±0.6 (n=70). In PDA media the mean growth of mycelia by 96h was 25 mm diameter (with 24h, darkness), and its appearance greyish-white to olive-gray and chlamy-dospores were not observed (Figure 1d). Although, those morphometric characteristics were similar to those reported for N. arbuti in chilean blueberries by Espinoza et al. (2009), Farr et al. (2005) indicated that N.arbuti has chlamydospores and L/W = [2.3-[3.1 ± 0.4 [-4.2] would be other characteristic to differentiating N. arbuti and N. nonquaesitum (Inderbitzin et al., 2010). Previous study performed on highbush blueberry (cvs. Brigitta and Elliott) reported morphometric characteristics similar to as Neofusicoccum nonquaesitum (Inderbitzin et al. , 2010).

3.3 Identification of the isolates and pathogenicity test

According to reported by CABI, the genetic analysis of obtained isolates was based on rDNA sequencing of ITS region, with total homologation (100%) to fungus N. nonquaesitum, available in the CABI collection under the number IMI-500168. Whereas, the sequencing was deposited into Genbank (accession number JX217819.1) based in the products ITS1 (1..177), 5.8S rRNA (178..334), ITS2 (335..488) and 28S rRNA (489..>524) (Table1).

Table 1. Neofusicoccum nonquaesitum strain CABI IMI-500168 internal transcribed spacer 1,5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence.

Pathogenicity test were positive in stems of both cultivars, observing a smooth discoloration of adjacent tissue after six days, symptoms were quickly developed, susceptibility varied between cultivars, by 21 day mean length of lesions was for Brigitta 15.5 cm and Elliot 4.9 cm (Figure 2), whereas control plants inoculated with sterile PDA plugs were healthy. The plants under stress conditions due to certain types of handling are frequently affected by Botryosphaeria spp., as a result of extensive occurrence and opportunistic nature of these species (Proffer and Jones, 1989; Caruso and Ramsdell, 1995). Spring and winter rains, abundant plant foliage, the size and form of conidia are related to dispersion of inoculum in field conditions (Ahimera et al., 2004). The infection produced by N. nonquaesitum is inversely proportional to tissue maturity, which is consistent with studies conducted on species of Botryosphaeria (Milholland and Meyer, 1984; Creswell and Milholland, 1987; Smith, 2009).

Figure 2. Pathogenicity test on control (left) and cankered stems (right) of cultivars Brigitta and Elliott.

After this study, has been frequently the detection of Neofusicoccum fungi, especially N. nonquaesitum and N. parvum, from different location sampling in La Araucania region. In fact, this study contributes to better understanding and consequently to an effective field control. Also is remarkable that La Araucania represent a 12% of the total V. corymbosum planted area in Chile.

4. Conclusions

The fungus Neofusicoccum nonquaesitum associated with twigs dieback and stem cankers in Brigitta and Elliott highbush blueberry cultivars, was identified according to morphological characteristics and genetic sequence, supported by CABI under number IMI-500168; detected in two commercial orchards in La Araucania and Los Rios regions, Southern Chile. The pathogenicity test demonstrated that Brigitta, compared with Elliott, was the most susceptibility to infection caused by N. nonquaesitum.

We think, in the future is necessary evaluating the incidence of this fungus in different varieties and local conditions. Also, is convenient to study in detail the complex species from Neofusicoccum genus reported in Chile.


Acuña, R. 2010. Compendio de Bacterias y Hongos de Frutales y Vides en Chile. Servicio Agricola y Ganadero. División de Protección Agricola y Forestal. 150p.         [ Links ]

Ahimera, N., Gisler, S., Morgan, D.P., Michailides, T.J. 2004. Effects of single-drop impactions and natural and simulated rains on the dispersal of Botryosphaeria dothidea conidia. Phytopathology. 94, 1189-1197.         [ Links ]

Agrios, G. 2005. Plant Diseases Caused by Fungi. In: Plant Phathology, Chapter11, 5th. ed, Elsevier Academic Press, USA. pp. 385-614.         [ Links ]

Alves, A., Phillips, A., Henriques, I., Correia, A. 2005. Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species. FEMS Microbiol. Lett. 245, 221-229.         [ Links ]

Baskarathevan, J., Jaspers, M., Jones, E., Ridgway, H. 2009. Evaluation of different storage methods for rapid and cost-effective preservation of Botryosphaeria species. New Zealand Plant Protection. 62, 234-237.         [ Links ]

Biggs, A.R., Britton, K.O. 1988. Presymptom histopathology of peach trees inoculated with Botryosphaeria obtuse and B. dothidea. Phyophathology. 78, 1109-1118.         [ Links ]

Caruso, F., Ramsdell, D. 1995. Compendium of Blueberry and Cranberry diseases. Edition APS Press. USA. 87p.         [ Links ]

Creswell, T., Milholland, R. 1987. Responses of blueberry genotypes to infection by Botryos-phaeria dothidea. Plant Dis. 71, 710-713.         [ Links ]

Crous, P., Slippers, B., Wingfield, M., Rheeder, J., Marasas, W., Phillips, A., Alves, A., Burgerss, T., Barber, P., Groenewald, J. 2006. Phylogenetic lineages in the Botryosphaeriaceae. Stud. Mycol. 55, 235-253.         [ Links ]

De Wet, J., Slippers, B., Preisig, O., Wingfield, B., Wingfield, M. 2008. Phylogeny of the Botryosphaeriaceae reveals patterns of host association. Mol. Phylogenet. Evol. 46, 116-126.         [ Links ]

Denman, S., Crous, P., Taylor, J., Kang, J., Pascoe, I., Wingfield, M. 2000. An overview of the taxonomic history of Botryosphaeria, and a reevaluation of its anamorphs based on morphology and ITS rDNA phylogeny. Stud. Mycol. 45, 129-140.         [ Links ]

Denman, S., Crous, P., Groenewald, J., Slippers, B., Wingfield, B., Wingfield, M. 2003. Circumscription of Botryosphaeria species associated with Proteaceae based on morphology and DNA sequence data. Mycologia. 95, 294-307.         [ Links ]

Espinoza, J., Briceño, E., Chávez, E., Úrbez-Torres, J., Latorre, B. 2009. Neofusicoccum spp. Associated with stem canker and dieback of blueberry in Chile. Plant Dis. 93, 1187-1194.         [ Links ]

Farr, D., Elliott, M., Rossman, A., Edmonds, R. 2005. Fusicoccum arbuti sp. nov. causing cankers on Pacific madrone in western North America with notes on Fusicoccum dimidiatum, the correct name for Scytalidium dimidiatum and Nattras siamangiferae. Mycologia. 97, 730-741.         [ Links ]

Guerrero, J. 1988. Enfermedades del arándano. In: Instituto de Investigaciones Agropecuarias, Estación Experimental Carillanca. El cultivo del arándano. Temuco, Chile. pp. 99-107.         [ Links ]

Guerrero, J. 1993. Situación fitopatológica de las especies fruticolas cultivadas comercialmente en la IX Región. Frontera Agricola. 1, 45-50.         [ Links ]

Guerrero, J. 2001. Situación fitopatológica del arándano alto (Vaccinium corymbosum L.) en la IX y X Región. Simiente. 71, 33-73.         [ Links ]

Inderbitzin, P., Bostock, R., Trouillas, F., Michailides, T. 2010. A six-locus phylogeny reveals high levels of species diversity in Botryosphaeriaceae from California almond. Mycologia. 102, 1350-1368.         [ Links ]

Larach, A., Besoain, X., Salgado, E. 2009. Crown and root rot of highbush blueberry caused by Phytophthora cinnamomi and P. citrophthora and cultivar sensitivity. Cien. Inv. Agr. 36, 433-442.         [ Links ]

McDonald, V., Eskalen, A. 2011. Botryosphaeriaceae Species Associated with Avocado Branch Cankers in California. Plant Dis. 95, 1465-1473.         [ Links ]

MycoBank. 2010. Available at http://www.mycobank. org/        [ Links ]

Milholland, R., Meyer, J. 1984. Disease and Arthropod pest of blueberries. North Carolina Agricultural Research Service, Bulletin 468. North Carolina, USA. pp33.         [ Links ]

ODEPA, 2013. Mercado y proyecciones del cultivo de arándanos. No 183, In: pdf        [ Links ]

Oliveira, V., Michereff, S., Brainer, R., Tuão, C., Gomide, E., Saraiva, M. 2010. Species of Botryosphaeriaceae associated on mango in Brazil. Eur. J. Plant Pathol. 127, 509-519.         [ Links ]

Perez, C., Altier, N., Simeto, S., Wingfield, M., Slippers, B., Blanchette, R. 2008. Botryosphaeriaceae from Eucalyptus and native Myrtaceae in Uruguay. Agrociencia. 12, 19-30.         [ Links ]

Pérez, S., Guerrero, J., Bensch, E. 2010. Botryosphaeria parva (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, y Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, asociados con muerte regresiva y cancros en arándano cv. Brigitta y Elliott, en la Región de La Araucania. In: XIX Congreso SOCHIFIT. Pucón, Chile.         [ Links ]

Phillips, A. 2010. The Botryosphaeria Site. Centro de Recursos Microbiológicos, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal. Available at http://www.crem.         [ Links ]

Prodorutti, D., Pertot, I., Giongo, L., Gessler, C. 2007. Highbush Blueberry: Cultivation, Protection, Breeding and Biotechnology. Eur. J. plant Sci. Biotech. 1, 44-56.         [ Links ]

Proffer, T.J., Jones, A.L. 1989. A new canker disease of apple caused by Leucostoma cincta and other fungi associated with cankers on apples in Michigan. Plant Dis. 73, 508-514.         [ Links ]

Rayachhetry, M., Blakeslee, G., Miller, T. 1996. Histopathology of Botryosphaeria ribis in Malaleuca quinquenervia: pathogen invasion and host response. Int. J. Plant Sci. 157, 219-227.         [ Links ]

Shenoy, B., Jeewon, R., Hyde, K. 2007. Impact of a DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Divers. 26, 1-54.         [ Links ]

Slippers, B., Crous, P., Denman, S., Coutinho, T., Wingfield, B., Wingfield, M. 2004a. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia. 96, 83-101.         [ Links ]

Slippers, B., Fourie, G., Crous, P., Coutinho, T., Wingfield, B., Wingfield, M. 2004b. Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea. Mycologia. 96, 1030-1041.         [ Links ]

Slippers, B., Smit, W., Crous, P., Coutinho, T., Wingfield, B., Wingfield, M. 2007. Taxonomy, phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world. Plant Pathol. 56, 128 -139.         [ Links ]

Úrbez-Torres, J.R., Peduto, F., Vossen, P.M., Krueger, W.H., Gubler, W.D. 2013. Olive twig and branch dieback: Etiology, incidence, and distribution in California. Plant Dis. 97, 231-244.         [ Links ]