DOI: 10.5061/dryad.p2ngf1vmx
title: Seasonal patterns of fungal colonisation in Australian native plants of different ages

description: Plant fungal relationships should vary with abiotic and biotic factors to
 minimise plant stress and are likely to vary seasonally and with age. We
 investigated how fungal colonisation, specifically arbuscular mycorrhizal
 fungi and dark septate fungal endophytes, would vary with species identity
 and season, and how these interactions change with ontogeny. Plant roots
 of adults and seedlings of 9 species were collected from heathland and
 coastal dune habitats along the Australian east coast in New South Wales.
 Roots were stained and investigated for arbuscular mycorrhizal fungi and
 dark septate endophyte structures to determine colonisation strength.
 Species identity was the most important factor driving colonisation
 strength, while low rainfall and heatwaves were associated with declining
 arbuscular mycorrhizal fungi colonisation in the warmest sampling period.
 AMF colonisation may be supressed by plants under heat and water stress as
 a way of avoiding loss of limited photosynthates. Dark septate endophyte
 colonisation was more common in this time period and may assist with the
 stress of the warmer, drier conditions. Colonisation by arbuscular
 mycorrhizal fungi differed with age but in unpredictable ways and, along
 with dark septate endophytes, was evident even in plants that are
 considered non-mycorrhizal, although more extensive in known mycorrhizal
 species. The lack of arbuscular mycorrhizal fungi colonisation and the
 increase in dark septate endophyte colonisation during the most stressful
 period suggest an uncoupling mechanism in the symbiotic relationship which
 needs further investigation.
description: Plant roots were collected from various locations of heathland
 and coastal dune habitats along the New South Wales (NSW) coast from the
 Sydney and the South East Coast bioregions. Sites spanned a distance of
 307 km from North Durras on the southern NSW coast (35°38′31.48”S
 150°18′11.11″E) to Ku-ring-gai Chase National park in Northern Sydney
 (33°35′55.71”S 151°17′33.62″E). Heathland plants were chosen as plants
 have adapted to withstand harsh conditions (Keith 2004) including skeletal soils low in nutrients with fire occurring periodically and often at high intensity (NSW Office of Environment and Heritage n.d.). Coastal dunes form an important ecological transition zone between marine and terrestrial environments where plants must overcome unstable sand, harsh winds, high stress from salt and drought, and limited nutrients (Hesp 1991). Species choice was first determined through the encounter of seedlings, followed by collection of adults from a different site. Families with specialised root structures such as proteoid roots were avoided as these are unlikely to have mycorrhizae (Brundrett 2008). Two species of Ericaceae were also collected to investigate EM.  Samples were collected over three time periods with different climatic variables; May – June 2017 was the coldest sample with temperature increasing from the second samples collected in the warmer months of August 2017 and the last samples collected between the hot months of December 2017 and January 2018. Average maximum and minimum temperature, and average rainfall were collected for the three time periods from Bureau of Meteorology (BOM) weather stations within the study range and compared to the expected averages for these time periods (Table 2). This study was interested in investigating broad patterns across time rather than site specific microclimates. Köppen Climate Classifications across the study region are Cfa (humid Subtropical) and Cfb (Oceanic) (Climate-data.org n.d.), as a result the temperature and rainfall was similar at each site within a sample period. Nine species were collected; each with three replicate plant samples collected for each life stage and for each season for a total of 162 samples. Adults and seedlings of the same species were collected from different locations each season, with at least one kilometre between sites to maintain independence of samples. Individual plants were separated by at least 50 m to increase spatial variability. Maximising distance between samples minimises the likelihood of sampling similar AMF communities and as this study was focused on larger landscape processes it is important to avoid local patterns on occurrence of fungi to be sure colonisation of a species was not confounded by local rhizosphere communities (Brundrett 2009). As soil microbe communities can vary over short distances, 50 m was considered far enough apart to maintain independence (Hazard et al. 2013; Huusko et al. 2017). Samples were collected by removing debris around the base of the specimen, digging soil away from the top 10–15 cm of the plant and following the taproot to finer root hairs which were collected. Seedlings were characterised by being less than 10 cm tall and lacking mature reproductive organs, while adults were characterised by being reproductively mature and at least 20 cm tall or long.   Fungal root colonisation was investigated using a mycorrhizal staining technique adapted from McGonigle et al. (1990). For each specimen, at least 15 thin root segments (&lt;2 mm diameter) were cut into 1 cm pieces and placed in 10% KOH in a 90<sup>o</sup> C water bath for 60 mins, after which they were rinsed with distilled water. More lignified root segments, such as the Ericaceae, required longer clearing and were placed in the bath for 10 min increments until sufficiently cleared or for a maximum time of 90 mins. Subsequently, the roots were covered with 1% HCl overnight and rinsed the following morning. Specimen jars were filled with 2% Quink in 1% HCl and placed in a 60<sup>o</sup> C bath for 30 mins to stain the roots. This solution was again rinsed from the roots and they were covered with a de-staining solution for three nights. This process acts to clear excess stain from the root allowing fungal tissue to be seen. At this point, 10 root segments of each specimen were mounted on a slide for investigation. For each slide the presence of fungal structures, both mycorrhizal and non-mycorrhizal, were calculated using a modification of the magnified intersections method outlined by McGonigle et al. (1990). Using a compound microscope at 40 x magnification, 100 root intersections on each slide were investigated. At each intersection, if the microscope crosshair touched a feature of AMF, EM or DSE its presence was recorded. AMF are characterised by aseptate hyphae, vesicles and arbuscules, while DSE are characterised by septate hyphae and microsclerotia (Likar et al. 2008; Bücking et al. 2012). EM are fungal endophytes characterised by fungal coils that form in fine root hairs (Chambers et al. 2008). For each AMF, DSE and EM structure, a sum was taken of each sample to provide a percentage (%) root colonisation of a given species.
description: This dataset shows for a given species, of a given ontogeny, at
 one of 3 time periods the % colonisation by AMF, DSE and Ericoid
 Mycorrhizal structures. There are no missing files.