Introduction

The ideal for foresters is to obtain high Eucalyptus seedling survival rates above 85% (Stape et al. 2001), but delay of seedling emergence and poor survival of seedlings remain a common nursery challenge. Several factors can reduce seedling emergence, among them is seed health status (Brown and Ferreira 2000; Lilja et al. 2010). In almost every harvested seed lot, chaff and other debris together with a variety of microorganisms are naturally present at least in small quantities (Boland et al. 1980). Seed-borne fungi can cause seed rot, delay seed germination or threaten establishment of plant stands due to pre- and/or post-emergence damping-off (Cram and Fraedrich 2010; Evira-Recuenco et al. 2015; Tobias et al. 2017). During processing or storage, infested seed batches may contaminate other clean seed lots (Agarwal and Sinclaire 1997).

Apart from seeds acting as primary sources of inoculum of diseases in nurseries, there is increased risk of spread of diseases across geographical borders through the seed trade (Elmer 2001; Santini et al. 2013). The rise in the seed trade in the last decades has increased the risk of spread of forestry pathogens such as Botryosphaeria dothidea (Moug.) Ces. & De Not., Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Mycosphaerella nubilosa (Cooke) Hansf. and Teratosphaeria zuluensis (M.J. Wingf., Crous & T.A. Cout.) M.J. Wingf. & Crous (Slippers et al. 2009; Hunter et al. 2011; Jimu et al. 2015; Maciel et al. 2015). In the last decade, different governments have passed tougher quarantine laws in trade of agricultural goods and services, but new pests and diseases continue to appear in Eucalyptus plantations (Graziosi et al. 2019). Hence, regular seed health tests are a prerequisite as decision-making tools for detecting and quantifying inoculum loads on seeds.

Although reports on seed-borne mycoflora associated with Eucalyptus have appeared from time to time (Mittal 1986; Farr et al. 1989; Mittal et al. 1990; Pongpanich 1990; Mehrotra and Singh 1998), most of these studies merely listed seed-borne mycoflora on a few Eucalyptus spp. without examining the effects of specific fungi on seed germination and seedling development. Jimu et al. (2015) investigated the mycoflora associated with Eucalyptus grandis W. Hill ex Maiden seed samples produced in South Africa, however the diversity of seed-borne mycoflora associated with various Eucalyptus species largely remains unknown. Therefore, the aim of this study was to investigate seed-borne mycoflora associated with commercial seeds of 12 different Eucalyptus spp., evaluate their effect on seed germination and use a detached leaf assay to determine their pathogenicity.

Materials and methods

Source of seed

One sample of each Eucalyptus spp. (Table 1), supplied by commercial forestry seed companies in South Africa, were used in this study. Seed lots were tightly sealed in plastic bags and stored at 4 °C until use.

Table 1 Incidences of fungi (%) associated with commercial Eucalyptus spp. seed lots produced in South Africa
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Seed health tests

Seed-borne mycoflora associated with Eucalyptus spp. seeds were investigated using the agar plate method. A weighed replicate (ISTA (International Seed Testing Association) 2018) of 0.1 g of each Eucalyptus spp. was wrapped in sterile cheesecloth and surface disinfected by soaking in 1% sodium hypochlorite solution for 5 min. After rinsing in sterile distilled water, seeds were spread out and air dried on sterile paper towels in a laminar flow. Ten seeds were plated in each 90 mm diameter Petri dish containing potato dextrose agar (PDA, Biolabs, Midrand, South Africa). Petri dishes were sealed with Parafilm® and transferred to a 25 °C incubator (Labcon growth chamber, Krugersdorp, South Africa). For each Eucalyptus species, four replicates of 10 Petri dishes were arranged in a completely randomised design. After 5 days of incubation, fungi growing from seeds were isolated, sub-cultured on PDA and incubated at 25 °C for 7 days under alternating cycles of 12 h near ultra violet (UV) (365 nm) light and darkness. Fungal genera and species were identified with the aid of various references of Ellis and Ellis (1997), Mathur and Kongsdal (2003) and Leslie and Summerell (2006). Incidences of seed-borne fungal species were determined by counting the number of times each fungal species appeared, and expressed as a percentage of seeds tested in each seed lot. Relative incidences of isolation of each fungal species were expressed as a percentage of the total number of fungal species observed in all four replicates. Fungal isolates were stored on PDA slants at 4 °C for further experiments.

Molecular identification

The molecular technique based on the Polymerase Chain Reaction (PCR) was used to confirm identity of selected seed-borne fungal isolates. From 7-day-old cultures, 100 mg of mycelium was scraped and DNA was isolated using Zymo DNA extraction kits (Zymo Research, USA) following the manufacturer’s protocol. Primer pairs ITS 1F and ITS 4R were used to amplify the Internal Transcribed Spacer (ITS1 and 2) conserved regions (White et al. 1990). Each 50-μl reaction mixture included 21 μL of PCR-grade water, 1 μL of DNA template, 1.5 μM of each primer, and 1 μL of PCR Master Mix (2X) (0.25 μL Taq DNA polymerase, reaction buffer, 4 mM MgCl2 and 0.4 mM of each dNTP; Thermo Scientific, Waltham, USA). The PCR conditions consisted of a denaturation step at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The amplified DNA was purified using a Zymo purification kit (Inqaba Biotech, South Africa), concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and adjusted to 50 ng/μL.

The purified PCR product was sequenced with PCR primers ITS 1F and ITS 4R and the BigDye terminator sequencing kit v.3.1 (Applied Biosystems, USA) with AmpliTaq® DNA Polymerase (Applied Biosystems, Warrington, UK). From forward and reverse sequences obtained, consensus sequences were compiled using BioEdit (www.mbio.ncsu.edu/BioEdit/BioEdit.html), and subjected to Blast searches in in GenBank [National Centre for Biotechnology Information (NCBI), (www.ncbi.nlm.nih.gov/BLAST)]. Fungal cultures were deposited in the National Collection of Fungi, ARC-Plant Health and Protection, Roodeplaat, Pretoria, South Africa and the respective sequences were deposited in GenBank at NCBI, (www.ncbi.nlm.nih.gov/genbank) (Table 2).

Table 2 Sequences recovered from fungi isolated from seed lots of Eucalyptus spp. matching sequences in NCBI GenBank
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Seed germination tests

The effect of 16 molecularly identified fungi isolated from Eucalyptus seeds (one isolate for each fungal species) on seed germination were evaluated for their effect on seed germination in -vitro. From 7-day-old cultures of each fungus, mycelia was scrapped and spores suspended in sterile distilled water amended with two drops of Tween 20 (Merck Ltd., Johannesburg, South Africa). The concentration of inoculum was adjusted to 1 × 105 spores/mL. Twelve Eucalyptus spp. seed lots, surface sterilised as described above, were inoculated with each of the sixteen fungi by soaking in 10 mL inoculum contained in a 150 mm glass Petri dish for 5 h at room temperature. Inoculated seeds were air dried on sterile paper towels in the laminar. Surface sterilised Eucalyptus seed lots soaked in sterile distilled water served as controls. Subsequently, seed germination was tested on four replicates of 50 inoculated and control seeds using the on-top paper method (ISTA (International Seed Testing Association) 2018). In each 150 mm glass Petri dish, 25 seeds were evenly spread out on top of two layers of moistened sterile filter papers (Whatman No. 1). Petri dishes containing plated seeds were incubated in a walk-in growth chamber (Seed Science Laboratory, University of Pretoria, South Africa). The plates received an alternating cycle of 10/14 h cool white light and darkness and temperature was maintained at 25 ± 1 °C. After 21 days, assessment of seed germination was done according to ISTA (International Seed Testing Association) (2018). Results of the experiment were scores of either healthy germinated seedlings without symptoms or diseased seedlings. Healthy germinated seedlings have intact primary roots and fully developed hypocotyls, whereas diseased seedlings were identified as those with necrotic spots or discolouration on the hypocotyl or seminal roots.

Seed-borne mycoflora pathogenicity assays

Pathogenicity assays were performed on detached leaves collected from 3-year old Eucalyptus plants grown in a nursery of the Forestry and Agricultural Biotechnology Institute (FABI, University of Pretoria, South Africa). Freshly collected, healthy looking leaves of E. benthamii, E. camaldulensis, E. dorrigoensis, E. dunnii, E. grandis, E. macarthurii, E. nitens, E. tereticomis, and E. viminalis were surface sterilized with 70% ethanol and rinsed thoroughly with sterile distilled water. Sixteen fungi isolated from Eucalyptus seed lots, listed in Table 2, were used. For each fungus, a 5 mm diameter mycelial plug of a 5-day-old culture was placed with the top side facing down on a sterilised leaf surface. Thereafter, inoculated leaves (three for each Eucalyptus sp.) were aligned on two layers of sterile moistened Whatman No.1 filter papers in glass Petri dishes. Inoculated Eucalyptus leaves were maintained in a walk-in growth chamber at 25 ± 1 °C. Control leaves were inoculated with 5 mm diameter agar plugs without fungi. Visual assessments of symptom development were recorded after five days of incubation based on relative size and colour of spots on inoculated leaves compared with non-inoculated controls. The experiment was repeated.

Data analysis

Results of germination tests were combined and subjected to analysis of variation (ANOVA) using SAS Version 9.4 statistical software (SAS Institute 2016), with the Fisher’s Least Significance Difference test (LSD, p = 0.05) separating significant differences between means. For pathogenicity tests, observations of infection of detached leaves were recorded in contrast with untreated controls.

Results

Seed health status

In this study, a total of 35 fungal species from 29 genera in addition to Penicillium species that was not identified to species level were found naturally associated with Eucalyptus seed lots. A total of 220 fungal isolates were obtained from Eucalyptus seed lots, among which 106 could be identified morphologically to the species level. The remaining 114 fungal isolates were left unidentified as fungi did not sporulate or produce other reproductive structures. The Eucalyptus nitens seed lot was the most infested, whereas the lowest incidence of fungi occurred on the E. dorrigoensis seed lot (Table 1). Taxonomic composition assessments showed a predominance by three genera: Penicillium, followed by Aspergillus and Alternaria. Genera rarely isolated in order of frequency included Stachybotrys, Ulocladium, Aureobasidium and Disculoides. Of the isolated fungi, confirmation of 16 seed-borne isolates exhibited high similarities with ITS sequences of reference isolates from GenBank (Table 2).

Seed germination tests

Percentage germinated seeds of the 12 Eucalyptus spp. inoculated with the 16 selected fungi are given in Table 3. Highest seed germination percentages were from non-inoculated seed lots, where E. dunnii, E. teritecomis and E. urophylla seed lots had percentages germination above 90%. However, seed germination was significantly reduced when seeds were inoculated with seed-borne fungi (p < 0.05). The lowest seed germination was recorded on E. badjensis (30.5%), E. benthamii (29.8%), E. dorrigoensis (37.0%), E. dunii (32.2%), E. grandis (37.0%), E. macathurii (28.3%), E. nitens (25.0%), E. pellita (30.5%), E. smithii (33.5%), E. tereticomis (31.8%), E. urophylla (31.3%) and E. viminalis (28.3%). On the contrary, inoculating Eucalyptus seed lots with S. polyspora and Chaetomium sp. had the least effect on seed germination. Germination was reduced the most by Botrytis sp. in E. benthamii and E. viminalis seed lots and by Colletotrichum in E. benthamii. Germination was most affected by Botrytis sp. in E. benthamii, E. dorrigoensis and E. grandis, F. oxysporum in E. nitens and F. solani in E. macathurii and E. nitens (Table 3).

Table 3 Percentage healthy seedlings from 12 Eucalyptus spp. seed lots inoculated with 16 selected fungi isolated from Eucalyptus spp.
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Seeds inoculated with seed-borne fungi yielded significantly higher numbers of diseased seedlings (p < 0.05) compared with non-inoculated controls which were naturally infested. The most diseased seedlings occurred in E. badjensis, E. benthamii, E. dorrigoensis, E. dunnii, E. pellita, E. smithii, E. tereticornis seed lots inoculated with either F. oxysporum (61.8, 55.8, 51.5, 57.8, 60.0, 55.0 and 57.5%, respectively) or F. solani. (60.8, 59.3, 53.0, 55.0, 57.5, 57.3 and 54.3%, respectively) when compared to their respective controls (Table 4). Similarly, inoculating E. benthamii, E. dorrigoensis, E. grandis, E. smithii and E. urophylla seed lots with Botrytis sp. yielded the most diseased seedlings (59.8, 52.3, 49. 0, 54.5 and 55.3%, respectively) when compared to their respective controls. Seedlings of E. benthamii were most susceptible to infection with either Botrytis sp. or Colletotrichum sp. E. nitens had highest disease susceptibility to F. oxysporum whilst E. macarthurii, E. nitens and E. urophylla were most susceptible to F. solani (Table 4).

Table 4 Percentage diseased seedlings from 12 Eucalyptus spp. seed lots inoculated with 16 selected fungi isolated from Eucalyptus spp.
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Seed-borne mycoflora pathogenicity assays

There were dark brown-black leaf spots on E. benthamii, E. camaldulensis, E. dorrigoensis, E. dunnii, E. grandis, E. macarthurii, E. nitens, E. tereticornis, and E. viminalis leaves inoculated with Disculoides sp., F. oxysporum, Lasiodiplodia sp. and Mycosphaerella sp. Inoculation with Botrytis sp., Botryosphaeria sp., F. solani, Phoma sp., Preussia sp., Nigrospora sp. or Ulocladium sp. produced light brown leaf spots on leaves of E. benthamii, E. dunnii and E. nitens. However, no leaf symptoms appeared on non-treated controls and Eucalyptus leaves inoculated with any of Aureobasidium, Chaetomium, Gliocladium and Sydowia species.

Discussion

Testing health status of seeds is essential for monitoring presence or absence of disease causing microorganisms that may affect seed germination and seedling development. Despite several countries implementing stricter phytosanitary regulations in the trade of agricultural products including live plants and seed (Cleary et al. 2019), phytosanitary requirements for most tree species, even the dominant tree species in commercial forest plantations, are minimal (Cleary et al. 2019).

Tree seeds are often infested with large numbers of fungi (Mittal 1986; Yuan et al. 1990; Mamatha et al. 2000; Sutherland et al. 2002; Cleary et al. 2019). This study showed that Eucalyptus seed lots were naturally infested with several fungi, where the highest incidence was recorded on E. nitens seed lot and the least on E. dorrigoensis. Variation of incidences of fungi on seed lots can be attributed to the influence of external environments of seed orchards but also different sources of possible contamination sites from harvesting to processing and storage (Cram and Fraedrich 2010). The season seeds are harvested and the level of maturity of capsules can influence the pattern of fungal richness isolated from seeds. Such variations are expected to be more pronounced due to morphological differences of seeds of species examined (Boland et al. 1980). Seed size, surface texture and shape are important characteristics that may influence the amount of fungi harboured in seed lots. Wrinkled seeds are more likely to harbour more pathogens than smooth surfaced seeds (Charkowski et al. 2001). This is particularly true for findings of this study, where fewer fungi were isolated from seeds of E. dorrigoensis and E. grandis as they have a uniform, more or less smooth, surface compared with more wrinkled and rough surfaced seeds of E. nitens (Boland et al. 1980).

The majority of fungi associated with seeds tend to have saprotrophic lifestyles with minimal negative effect on seed germination and seedling growth. A total of 29 fungal genera were found naturally associated with Eucalyptus seed lots. Taxonomic composition assessments showed that Eucalyptus seeds were predominantly infested with saprotrophs, Penicillium (49.9%), Aspergillus (8.1%) and Alternaria (7.4%), which have been previously reported to cause significant reduction of Eucalyptus seed germination and seedling emergence (Doshi et al. 1993; Yuan et al. 1997). Moreover, due to their fast growing saprotrophic characteristic, slow growing fungi were inhibited and obscured. In general, many pathogenic fungi are characterised by slow growth on media, such as Teratosphaeria, taking more than 4 weeks to reach a diameter of 40–50 mm (Cortinas et al. 2006). Since isolations of fungi in this study were done using the culture based approach, estimates of fungal incidence in this study were conservative as several isolates were left unidentified due to lack of sporulation. Although isolations on media is cheap, it is limited and often fails to detect certain fungal groups such as basidiomycetes that seldom produce asexual or sexual spores in culture upon which identification is based.

The trade of seed carries with it risks of inadvertent introduction of pests and pathogens to previously unaffected regions. The majority of seed-borne fungi such as Lasiodiplodia, Neofusicoccum and Mycosphaerella found on commercial seed lots are already widely distributed geographically and do not pose a significant quarantine threat. However, there is a quarantine concern as this study reports first occurrences of Aureobasidium pullulans and Disculoides eucalypti on Eucalyptus seeds. The genus Disculoides was described in 2012 with D. eucalypti and Disculoides eucalyptorum Crous, Pascoe, I.J. Porter & Jacq. Edwards, being isolated from diseased E. viminalis leaves in Australia (Crous et al. 2016). In New Zealand, Disculoides eucalypti Crous, Pascoe, I.J. Porter & J. Edwards, intercepted on imported Eucalyptus leucoxylon F. Muell., was short-listed as a quarantine threat to the country’s biodiversity (Surveillance 2016; Crous et al. 2016). Detection of Botryosphaeria dothidea (Moug. ex Fr) Ces. & De Not on commercial Eucalyptus seeds is of quarantine significance as it appears on the European and Mediterranean Plant Protection Organization (EPPO) database of quarantine pests (https://gd.eppo.int/taxon/BOTSDO).

Seeds infected or contaminated with fungi may be damaged and fail to germinate, produce weak seedlings or may develop diseases on seedlings. Findings of this study showed that germination of Eucalyptus seed lots inoculated with seed-borne fungi resulted in a wide range of symptoms that included rotting of seeds, formation of lesions on newly developed hypocotyls and seminal roots or abnormal twisting of germinated seedlings. After inoculation, seed germination was less than 62% and as low as 25%, which potentially translates to low chances of seedling survival in nurseries. However, occurrence of diseased seedlings from non-inoculated controls suggest the presence of natural infection as confirmed by seed health tests. Botrytis and Fusarium spp. inoculated seed consistently yielded the lowest percentage of healthy seedlings on all Eucalyptus species. The notoriety of Fusarium as a serious threat to seedling emergence in numerous forest nurseries is well documented (Omokhua et al. 2009; Gordon et al. 2015; Won et al. 2019). The pathogen is a persistent problem in nurseries as it can cause severe pre- and post-emergence damping-off, and mortality of mature trees in forest plantations. In the seed lot samples examined in this study, soil-borne pathogens such as Fusarium oxysporum and F. solani might have been introduced on seeds at harvesting as capsules often fall on the ground of seed orchards. Thus, the impact of superficial contamination on seed germination and subsequent seedling damage in nurseries at a later stage is not to be underestimated.

In -vitro assays showed that inoculum of seed-borne A. alternata, B. dothidea, C. globosum, C. brachyspora, P. curvatum, D. eucalypti, L. theobromae, N. sphaerica and P. africana did not only reduce seed germination percentages but were also pathogenic on detached leaves of Eucalyptus. Although the leaf detached assay is a fast means of evaluating pathogenicity and severity of fungi, in -vitro detached leaves and plantlets are more susceptible than intact leaves of plants in the greenhouse or field (Townley et al. 2001; Liu et al. 2007).

In conclusion, findings of this study showed a large diversity of fungi associated with commercial Eucalyptus seed lots, some of which were pathogenic in a detached Eucalyptus leaf assay, and many reduced seed germination of Eucalyptus seed lots. The importance of the seed health and testing of Eucalyptus seed lots has been highlighted.