Calonectria ilicicola, Gliocladiopsis sp. and Ilyonectria liriodendri were isolated from diseased roots of young avocado trees. Pathogenicity studies with seedlings of three avocado cultivars, Velvick, Hass and Reed, demonstrated that Calonectria ilicicola is a severe root rot pathogen, reducing the biomass of healthy roots, and reducing plant height over time. Calonectria ilicicola was re-isolated from diseased roots. Ilyonectria liriodendri and Gliocladiopsis sp. were not pathogenic and plant height was increased after Gliocladiopsis sp. amendment compared to all other treatments in trials with cvs Velvick and Hass.
Fungi in the Nectriaceae (Hypocreales, Ascomycota) are well known pathogens of many agricultural and forestry crops worldwide, and taxonomic and pathological data have been extensively reviewed and revised (Crous, 2002; Lombard et al., 2010; Chaverri et al., 2011). They can cause a wide range of symptoms including damping-off, root rot, stem lesions, branch and crown cankers, leaf and shoot blight and fruit disease (Crous, 2002). Care is needed when considering the plant pathology literature for many nectriaceous fungi, as identifications made since the adoption of molecular phylogenetic species criteria may not correspond to morphologically based species.
In recent years, several isolates reported as Calonectria, Cylindrocladiella and Neonectria were obtained from the diseased roots of young avocado trees that had declined or died soon after transplanting into orchards in Australia. The literature on the pathogenicity of these fungi in avocado is limited, with most reports concentrating on Cylindrocarpon sp., the earlier anamorphic name for Neonectria and Ilyonectria.
Cylindrocarpon sp. was one of many fungi isolated from feeder roots of avocado trees exhibiting symptoms of tree decline (chlorotic or brown leaves, leaf drop, tree death) between 1986 and 1988 in Spain (Lopez-Herrera & Melero-Vara, 1992). Subsequent pathogenicity tests with 6-month-old avocado cv. Topa Topa plants indicated that Cylindrocarpon and Fusarium spp. were frequently reisolated from necrotic roots, but did not cause severe above-ground symptoms. The authors suggested that these fungi reduced tree vigour and predisposed trees to infection by other fungal or oomycete pathogens, such as Rosellinia necatrix and Phytophthora cinnamomi.
Similarly, a survey of all avocado nurseries in Israel was initiated following observations of dieback and severe root rot of young trees heavily laden with fruit, 2–5 years after planting in the orchard (Zilberstein et al., 2007). Cylindrocarpon sp. was isolated at frequencies of 10–100% from roots of apparently healthy seedlings. The fungus was confirmed as Neonectria radicicola (anamorph Cylindrocarpon destructans) based on morphological characterization and homology of small subunit ribosomal DNA sequences to those on genetic databases (Zilberstein et al., 2007). Cylindrocarpon destructans was also consistently isolated from commercial nursery-grown avocado plants with black root rot in Chile (Besoain & Piontelli, 1999) where it caused significant losses, estimated at 22 000 nursery trees during 1994 and 1995. Avocado plants with black root rot wilted, had marginal leaf chlorosis and necrosis, and often died suddenly. Contaminated water was shown to be the source of the pathogen.
In South Africa, Darvas (1978) isolated Cylindrocarpon destructans and Cylindrocladium parvum (synonym Cylindrocladiella parva) and less often Cylindrocladium scoparium (synonym Calonectria morganii), from avocado roots and from the root zone. Crous (2002) and Lombard et al. (2010) noted that Calonectria morganii was restricted to North America, Europe and Brazil and that all South African and Australian strains examined were Calonectria pauciramosa. Pathogenicity tests showed that by 7 days after inoculation of large feeder roots, each one of these three fungi killed 35–62% of the inoculated avocado roots (Darvas, 1979). Darvas (1979) did not mention whether the roots from which he originally isolated these fungi were diseased, or whether the associated trees were unhealthy.
There are three specimens of nectriaceous fungi associated with root diseases of avocado in Queensland held in the DEEDI Plant Pathology collection. In 1972, a Cylindrocladium sp. (BRIP 4405a) was found sporulating profusely on avocado leaf lesions at Levers Plateau, Rathdowney. In 1981, Cylindrocladiella parva (as Cylindrocladium parvum, BRIP 13522a), was associated with the death of 3-year-old cv. Wurtz trees at Woombye. The third and more recent specimens of nectriaceous fungi were isolated from avocado roots of a 5-month-old tree with dieback that was planted in an orchard 140 km north west of Brisbane. Approximately 50% of the root sample had severe root rot symptoms. There are no records of pathogenicity testing of these isolates in avocado or any other host.
The current study was conducted to confirm identity of the fungi that had been recently isolated from diseased roots of avocado nursery trees, and to establish pathogenicity of three of these fungi.
Materials and methods
Initial isolation of fungi
A selection of nine young vegetatively cloned avocado rootstocks and seedlings collected from a commercial nursery were examined. The plants were chosen due to their apparent ill-thrift compared to others, but none had severe foliar dieback. The root systems of most samples were severely necrotic with very few healthy white feeder roots. Root tissue was surface sterilized in 70% ethanol, washed in sterile distilled water and plated out on potato dextrose agar amended with streptomycin (sPDA). Cultures were examined for fungal growth, subcultured onto sPDA, and tentative identifications made based on colony colour and macroconidia shape and size.
Calonectria sp. (Cylindrocladium sp.) alone was the most frequently isolated (from five of nine plants). Ilyonectria (Cylindrocarpon-like sp.) alone was isolated from a single plant. One plant had mixed infections of Calonectria sp. and Gliocladiopsis sp. and two plants had mixed infections of Calonectria sp. and Ilyonectria sp., with one of these plants also being infected with Phytophthora cinnamomi. Single germinated conidial isolates were established and single isolates of Calonectria sp., Gliocladiopsis sp. and Ilyonectria sp. lodged in the culture collection of Herbarium BRIP at the Ecosciences Precinct, Dutton Park, Queensland, with accession numbers BRIP 53653, 53654 and 53652, respectively. These isolates were subsequently used in pathogenicity tests.
Preparation of inoculum and amendment of planting mix at seedling transplantation
Flasks (500 mL) were filled with 300 g of a sand:bran:water mixture (10:1:4 w/w) and autoclaved (Crous, 2002). Two plugs from the margins of fungal colonies actively growing on sPDA were inoculated into the media, and maintained on the laboratory bench at 24°C for 7–8 days, with shaking each day to ensure even colonization of the media.
For Trials 1 and 2, with cvs Velvick and Hass seedlings, respectively, the colonized media was added to equal volumes of fine vermiculite and thoroughly mixed. This inoculum mix was placed in the lower 5 cm of plastic pots (8 cm diameter, 16 cm height), and a single seedling planted using Searle’s premium potting mix for fill. The unamended control pots received uncolonized media/vermiculite mix.
In Trial 3, with cv. Reed seedlings, pots for transplantation were prepared by one-third filling with potting mix, then amending with four mycelial plugs per pot of the test fungus (or sterile sPDA as the unamended control). Seedlings were then transferred and pots filled with potting mix.
Pots were kept in controlled environment cabinets or glasshouses/polyhouses maintained at approx. 22–24°C day, 18°C night, watered daily with dechlorinated water and all plants fertilized with Thrive concentrate all purpose plant food (Yates Australia, N:P:K 12·4:3:6·2) as required.
Trial 1 – Velvick seedlings
Test fungi (or sterile media) were introduced to the pots as the seedlings were transplanted on 18 August 2010. There were 10 seedlings for each of the three test fungi (treatments) and unamended control. Plant heights above ground were measured at 3 and 4 weeks, and at 14 weeks plant heights were measured before uprooting seedlings, and root systems examined visually for percentage of healthy roots. Fresh root pieces from four plants per treatment were plated onto sPDA (eight pieces per plant), and incubated in the dark at 24°C, and examined for fungal growth. Root mass was determined after severing the roots below the seed, washing to remove potting mix, and weighing after drying at 60°C.
Trial 2 – Hass seedlings
Test fungi (or sterile media) were introduced to the pots as the seedlings were transplanted on 13 September 2010. There were 10 seedlings for each of the three test fungi (treatments) and unamended control. Plant heights above ground were measured at 4 days, 4 weeks, 6 weeks and 10 weeks, and at 19 weeks plant heights were measured again before uprooting seedlings and root systems examined visually for percentage of healthy roots. Root mass was determined as described above.
Trial 3 – Reed seedlings
Test fungi (or sterile media) were introduced to the pots as the seedlings were transplanted on 24 February 2011. There were 15 seedlings for each of the three test fungi (treatments) and unamended control, except for Gliocladiopsis sp. where there were 14 seedlings. Plant heights above ground were measured at 1 and 4 weeks, and at 6·5 weeks plant heights were measured again before uprooting seedlings and root systems were examined visually for percentage of healthy roots. Fresh root pieces from every plant per treatment were surface sterilized and plated onto sPDA (eight pieces per plant), and cultures maintained in the dark at 24°C, and examined for fungal growth. Root mass was determined as described above.
Cultures of Calonectria, Ilyonectria and Gliocladiopsis that were reisolated from diseased roots in this trial were lodged with the DEEDI culture collection, and re-assigned accession numbers BRIP 54018 (from plants inoculated with BRIP 53653), 54020 (from plants inoculated with BRIP 53652) and 54019 (from plants inoculated with BRIP 53654), respectively. These were subjected to molecular and morphological characterization to confirm identity.
Plant height, percentage of healthy roots and root dry weight data for each experiment were analysed separately by anova, and comparisons between means were made using Fisher’s protected LSD test.
Cultural characteristics were observed after 14 days growth on PDA in the dark at 24°C. Morphological characteristics were observed after 7–10 days growth on carnation leaf agar (CLA) in the dark at 24°C. Images of fungal structures were taken with a Leica DM2500 compound microscope with a Nomarski interference condenser.
Mycelia from isolates were placed in 2·0 mL safe-lock tubes (Eppendorf), then 0·5 mm glass beads (Daintree Scientific) were added and the mycelium was lysed using a Tissue Lyser (QIAGEN) for 2 mins at 30 hz s−1. DNA was extracted from this mixture using the Gentra Puregene kit (QIAGEN), following the manufacturer’s instructions. PCR amplification was conducted using the Mango Taq PCR Master Mix (Bioline), which consisted of 12·5 μL of 2 × Master Mix, 0·5 μL each of 10 mm forward and reverse primers, and 1 μL of DNA template. The internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (White et al., 1990), part of the β-tubulin-2 (BT) gene was amplified with primers T1 (O’Donnell & Cigelink, 1997) and CYLTUB1R (Crous et al., 2004), and part of the histone 3 (H3) gene was amplified with primers CYLH3F and CYLH3R (Crous et al., 2004). PCR products were amplified in a Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories) using the following conditions: 95°C for 2 min, 30 cycles at 95°C for 30 s, 55°C (for ITS) or 52°C (for BT and H3) for 30 s, 72°C for 1 min, followed by a 5 min final extension at 72°C. The products were purified using the QIAquick PCR Purification Kit (QIAGEN). Purified PCR products were sequenced by Macrogen Incorporated using the AB 3730xl DNA Analyser (Applied Biosystems). The sequences obtained were compared to the nucleotide sequence database (GenBank) using the nucleotide query blastn accessed via the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/).
Trial 1 – Velvick seedlings
There were significant differences (P < 0·005) among seedling heights at each measuring interval. At 3, 4 and 14 weeks after transplanting, average plant height from Calonectria sp. amended pots was significantly less than uninoculated controls and Gliocladiopsis sp. treatments (P <0·01). At 14 weeks after transplanting plants from Gliocladiopsis sp. amended pots were significantly higher, compared to all other treatments (P <0·001; Table 1).
Table 1. Avocado cv. Velvick seedling heights, root dry weights and % healthy roots after amendment of potting mix with test fungi
Amendment to potting media
Seedling height (cm)a
% healthy roots
Root dry weight (g)
aMean values within columns followed by the same letter are not significantly different at P =0·05.
Roots from plants receiving Calonectria sp. showed large areas of severe dark brown or black necrosis, and also smaller brown necrotic lesions, 14 weeks after amendment (Fig. 1). The percentage of healthy roots at this time was greatest in plants from pots amended with Gliocladiopsis sp., and was significantly different from those which had been amended with Calonectria sp. or Ilyonectria sp. (P <0·01; Table 1). Roots were the least healthy from plants receiving Calonectria sp. at transplanting. Although there were no significant differences among treatments, dry root weights were greatest from plants which received uncolonized media (uninoculated controls) or Gliocladiopsis sp. Root mass was the least in plants from Calonectria sp. amended pots (Table 1).
No pathogens were isolated when root pieces from four plants of the unamended control treatment were plated onto sPDA. Calonectria sp., Gliocladiopsis sp. and Ilyonectria sp. were reisolated from roots of plants from their respectively-amended transplant mix at frequencies of 66%, 69% and 32%, respectively.
Trial 2 – Hass seedlings
There were no significant differences among treatments in plant heights at the first three measuring times, 4 days and 4 and 6 weeks after amendment with test fungi. However, 10 and 19 weeks after amendment with Calonectria sp., plants were significantly smaller than those from all other treatments (P <0·001). At 19 weeks plants from the Gliocladiopsis sp. amended pots were significantly taller than plants from all other treatments (Table 2). The percentage of healthy roots and the dry weight of roots was significantly less from plants amended with Calonectria sp. than those from all other treatments at the final assessment, 19 weeks after amendment of potting mix with the test fungi (P <0·01; Table 2).
Table 2. Avocado cv. Hass seedling heights, root dry weights and % healthy roots after amendment of potting mix with test fungi
Amendment to potting media
Seedling height (cm)a
% healthy roots
Root dry weight (g)
aMean values within columns followed by the same letter are not significantly different at P = 0·05.
Trial 3 – Reed seedlings
This trial was shorter in duration than those with cvs Velvick and Hass and there were no statistically significant differences among treatments in seedling heights at any of the assessment times, although plants from Calonectria sp. amended pots were the smallest and were about 13% shorter than the control pots at 6·5 weeks after amendment of the potting mix. There were no significant differences in dry weights of above-ground parts or roots. However, roots from Calonectria sp. amended pots were significantly less healthy than those from all other treatments, 6·5 weeks after amendment (P <0·001; Table 3).
Table 3. Avocado cv. Reed seedling heights, root dry weights and % healthy roots after amendment of potting mix with test fungi
Amendment to potting media
Seedling height (cm)a
% healthy roots
Dry weight foliage (g)
Dry weight roots (g)
aMean values within columns followed by the same letter are not significantly different at P = 0·01.
Roots from all 15 plants per treatment (14 plants in Gliocladiopsis sp.) were plated out for confirmation of Koch’s postulates. No pathogens were isolated from roots of plants where sterile agar plugs were placed into pots when seedlings were transplanted. Calonectria sp. was isolated with high frequency (89% of total root pieces plated) from roots of all plants where pots had been amended with that fungus. In one plant Calonectria sp. was also isolated at a low frequency (1·7% of total root pieces plated). Gliocladiopsis sp. was isolated from roots of all plants where pots had been amended with that fungus. Of the total root pieces plated, 53·6% grew Gliocladiopsis sp.; Ilyonectria sp. was recovered less frequently. One root piece plated from each of six plants grew colonies of Ilyonectria sp., representing a frequency of isolation of 5%. However, no other fungal pathogens were isolated from these roots.
Molecular and morphological characterization
The BT and H3 sequences of BRIP 53653 and 54018 were 99% similar to the ex-type culture of Calonectria ilicicola CBS 190.50. The ITS and BT sequences of BRIP 53652 and 54020 were identical to the ex-type culture of Ilyonectria liriodendri CBS 110.81. The sequences of 53654 and 54019 had no direct match to any Nectriaceae in GenBank, but were confirmed by Dr L. Lombard to represent a new species within the Gliocladiopsis tenuis complex. (Dr L. Lombard, University of Pretoria, South Africa, personal communication.) All sequences have been deposited into GenBank and the accession numbers are listed in Table 4.
Table 4. GenBank accession numbers of isolates from this study and relevant references
The identity of BRIP 54018 as C. ilicicola was confirmed by morphological examination of colonies on PDA after 14 days in the dark (Fig. 2). These colonies covered the entire surface of the plate, were rust coloured (surface and reverse) with sparse aerial mycelium; and speckled in reverse due to abundant darker chlamydospores in the agar. Perithecia were scattered and abundant on the surface of the agar, globose to ellipsoidal, 300–500 μm diameter, orange to red. Asci were 8-spored, clavate, 75–120 × 11–17 μm; with ascospores in the upper part of the ascus. Ascospores were hyaline, fusoid, slightly curved, mostly 1-septate 33–58 × 5–6·5 μm. Conidia and conidiophores did not form on CLA agar after 7 days in the dark. A more detailed description of C. ilicicola can be found in Crous (2002). Calonectria ilicicola has a wide host range with many records in the literature appearing under its anamorphic synonym Cylindrocladium parasiticum (Crous et al., 1993).
The identity of BRIP 54020 as I. liriodendri (Chaverri et al., 2011) was confirmed by morphological examination of colonies on PDA after 14 days in the dark (Fig. 2). These colonies covered the entire surface of the plate and were cinnamon (surface and reverse) with sparse aerial mycelium. On CLA agar after 7 days in the dark, conidiophores were simple or sparsely branched, septate or consisting of only the phialide; phialides, 25–65 × 2·5–3·5 μm, widest at the base. Macroconidia were abundant on leaf pieces, 1–3 septate, cylindrical, 3-septate conidia 28–62 × 5–7·5 μm, hyaline. Microconidia were abundant on the agar surface, 0–1 septate, cylindrical to broadly ellipsoidal, 7–17 × 3–4·5 μm, hyaline. Ilyonectria liriodendri has synonyms in Neonectria liriodendri (Chaverri et al., 2011) and Cylindrocarpon liriodendri (Macdonald & Butler, 1981).
The identity of Gliocladiopsis sp. (BRIP 54019) was confirmed on the basis of morphology (Fig. 2). Colonies on PDA after 14 days in the dark were 5 cm in diameter, cottony, raised, upper surface hazel with irregular rings of white cottony mycelium around the margin and near the centre, with smooth to undulating margins, reverse chestnut and paler towards the margin. On CLA agar after 7 days in the dark, conidiophores were penicillate, mononematous and hyaline; conidiophores comprised a stipe and a penicillate arrangement of fertile branches; stipe 50–125 × 5–9 μm, septate, hyaline, smooth; penicillate conidiogenous arrangement branched, primary branches septate or aseptate up to 55 μm long; secondary and tertiary branches aseptate, up to 17 and 12 μm long respectively, each terminal branch producing up to four phialides; phialides cylindrical, hyaline, 10–15 × 1·5–2·5 μm. Subverticillate conidiophores sparse, comprising a septate stipe and primary and secondary branches up to 20 and 30 μm long respectively, terminating in 2–4 phialides; phialides cylindrical, hyaline, 13–33 × 2–3 μm. Conidia cylindrical, straight, medianly septate, 14–18 × 1·5–3·0 μm. This genus is morphologically similar to Cylindrocladiella but differs in lacking the stipe extension (Crous, 2002; Seifert et al., 2011).
This study has demonstrated that C. ilicicola is a severe root rot pathogen of young avocado trees. The fungus consistently reduced the portion of healthy roots compared to controls and the other test fungi in separate trials with three cultivars. In two trials, amendment of potting media with this fungus impacted negatively on plant heights over time, and root biomass. Calonectria ilicicola could be reliably reisolated from diseased roots, fulfilling the requirements of a pathogen according to Koch’s postulates. This is the first report demonstrating pathogenicity of C. ilicicola on avocado.
Calonectria ilicicola has a wide international distribution and host range. It causes peg, pod and root necrosis of peanuts (Bell & Sobers, 1966), and red crown rot in soybean (Berggren & Snow, 1989). Studies in those species have demonstrated that severity of disease may be affected by cultivar susceptibility, soil temperature and host age (Kuruppu et al., 2004). The pathogen also causes collar rot of papaya seedlings and young replants (Nishijima & Aragaki, 1973), and pathogenicity testing demonstrated differential susceptibility among cultivars, although a variety more tolerant in greenhouse screening tests suffered significant losses in commercial orchards in Hawaii (Nishijima & Aragaki, 1973). The papaya isolate was also extremely pathogenic on three species of Acacia, peanut and several Eucalyptus spp., demonstrating its lack of host specificity (Nishijima & Aragaki, 1973).
Ilyonectria liriodendri was not pathogenic to avocado roots in the current study. Previously known as Cylindrocarpon liriodendri or Neonectria liriodendri, it has been established as the causal agent of black foot disease, a root rot, of grapevines in many parts of the world, including Australia (Whitelaw-Weckert et al., 2007), South Africa (Halleen et al., 2006) and California (Petit & Gubler, 2007). It has not previously been reported from avocado, and it was most likely isolated as a ubiquitous rhizosphere inhabitant, and may have some degree of host specificity for grapevine. The recommended management for black foot disease in grapevine nurseries is to subject roots of dormant vines to hot water, 50°C for 30 min, followed by 30 min in cold water (Halleen et al., 2007). Cylindrocarpon destructans (=Ilyonectria radicicola) represents a complex of fungi from which I. liriodendri was segregated (Halleen et al., 2006). There are reports of isolates of Cylindrocarpon destructans as pathogenic on avocado (Darvas, 1979; Besoain & Piontelli, 1999; Zilberstein et al., 2007). Further surveys for nectriaceous pathogens associated with dieback of avocado are needed to resolve their pathogenicity and taxonomic status.
The isolate of Gliocladiopsis sp. used in these trials was not pathogenic to roots. Plant heights in two trials were significantly enhanced by amendment with Gliocladiopsis sp. compared to plants growing in uncolonized media.
Clean planting material is the most critical step in successful prevention of black root rot disease, caused by C. ilicicola, in avocado. Although nursery trees may look healthy, root examination followed by rapid diagnosis will quickly determine whether this pathogen, or another insidious root pathogen such as Phytophthora cinnamomi, is present. If C. ilicicola is introduced into an orchard, it will be difficult to eliminate as it produces microsclerotia in the host root tissue which serve as survival and dispersal structures and can persist for several years in soil or host debris. Hot water treatment of roots could be tested but is unlikely to be as successful as control of Cylindrocarpon liriodendri in grapevine (Halleen et al., 2007) as avocado has no dormant stage and is sensitive to hot water treatment. Further work on cultivar susceptibility, temperature and soil moisture conditions favouring infection and disease development in avocado is required.
The avocado disease management projects, AV07000 and AV10001, have been funded by the Australian Federal Government through its agency Horticulture Australia Ltd with levy support from Avocados Australia Ltd.