The potential for vesicular-arbuscular mycorrhizal fungi to influence the recovery of Hawkesbury sandstone vegetation after disturbance

Disturbance to plant communities by agricultural and mining practices is an inevitable intrinsic part of civilization. Growing public, political and scientific concern about problems such as land degradation and the greenhouse effect is increasing interest in revegetation as a counter-measure. One of the primary stresses imposed upon seedlings colonizing disturbed sites is a lack of nutrients. Plants species colonized by vesicular-arbuscular mycorrhizal (VAM) fungi have higher tissue levels of some inorganic nutrients (especially phosphorus), greater biomass yield, and more rapid uptake of water, and they are often more tolerant of various forms of stress than non-mycorrhizal plants of the same species. Consequently, it has been hypothesized that the successful revegetation of disturbed plant communities may be dependant in part on the availability of viable mycorrhizal inoculum. The studies described in this thesis were aimed at assessing the potential for VAM fungi to influence the revegetation of disturbed plant communities. VAM fungi were studied in detail in two quite different plant communities on Hawkesbury Sandstone soils in southeastern New South Wales. Although the sites supported vegetation of different physiognomy, 14 plant species were common to both sites. The following features ofthe ecology of VAM fungi are presented in this thesis: (i) the mycorrhizal associations of plant species in the two study sites, (ii) an examination of which part of an intact soil profile represents the major store of potential propagules of VAM fungi, (iii) an investigation of the relationship between the intensity of topsoil disturbance and the infectivity of VAM fungi, and (iv) an examination of which propagules of VAM fungi are capable of initiating VAM after topsoil disturbance. At both sites, an assessment of the mycorrhizal status of each of the plant species was made. Roots from five representatives of each plant species were collected from random locations within both of the study areas during July-September 1989. Fine feeder roots were assayed for mycorrhizal infection. At the woodland site, 21 of the 32 plant species examined had mycorrhizal associations. At the shrubland site, 31 of the 47 plant species examined were mycorrhizal. Internal hyphae, vesicles, and cortical hyphal coils were discovered on the roots of two species of Cyperaceae and on the non-proteoid roots of nine species of the Proteaceae. Several species within genera and families previously known to be mycorrhizal were also found for the first time to have associations. Endomycorrhizal associations predominated at both sites, but several species had both ecto- and endomycorrhizal associations. The presence or absence of mycorrhizal associations was consistent in those plant species common to both sites. The formation of VAM in intact soil profiles was measured in topsoil and subsoil using bioassay seedlings grown in intact soil cores. VAM most readily developed in the roots of bioassay seedlings grown in the topsoil. Limited VAM occurred in the roots grown in subsoil cores. Most colonization of roots by VAM fungi occurred in the soil cores collected and assayed during Spring and Summer. Few spores were found in any soil sampled, though at least twice as many spores occurred in the topsoil than in the subsoil, for all seasons examined. As most of the propagules that could initiate VAM (i.e. spores, colonized root fragments and fungal hyphae), were observed in the topsoil, disturbances which involve the removal and storage of the top 15 c m will adversely affect these fungi. Removal and storage of the surface layers of soil is known to decrease the infectivity VAM fungi. In previous studies investigating VAM fungi and soil disturbance, only two treatments have been examined viz. no disturbance v. profoundly disturbed soil. I investigated the relationship between increasing intensity of topsoil disturbance and the infectivity of VAM fungi. Intact soil blocks were treated with one of four levels of disturbance. Seeds of a bioassay species were sown into the blocks and harvested 14, 21, 28, 35, and 42 days after sowing. Colonization of roots by VAM fungi had commenced by 14 days in the intact, low, and intermediate disturbance treatments. Colonization of roots was delayed by up to six weeks for seedlings grown in the most disturbed of the soil blocks. Although the low and intermediate degrees of soil disturbance did not cause a delay in the initiation of infection, they did reduce the proportion of root length colonized by VAM fungi after 21 days. After 21 days, shoot biomass was significantly less in seedlings grown in the most disturbed of the soil blocks. The most severe experimental treatment probably disturbed the external hyphal network and the infected root fragments (containing VAM hyphae and vesicles), which in turn temporarily reduced the infective potential of the fungus to nil. The observed delay in the initiation of infection could therefore be explained by the time required for hyphae to grow from other propagules in the soil. The propagules of VAM fungi include: (i) spores, (ii) root fragments containing VAM hyphae and vesicles, and (iii) soil hyphae. The viability of each type of propagule after soil disturbance will determine in part, the number of infective propagules available to initiate the VAM association with plants re-colonizing a disturbed site. The aim of this study was to examine which of the propagules of VAM fungi are capable of initiating VAM after topsoil disturbance. Soil from the open woodland site was wet-sieved through a tier of three sieves (1 mm , 250 μm, and 106 μm), and separated into: (i) root fragments, (ii) VAM hyphae, (iii) VAM spores. Each of these fractions was assayed to determine its potential to initiate VAM . Fungal hyphae grew from root fragments after 14 days. VAM hyphal fragments did not produce any VAM infection even after 42 days. The VAM spore fraction initiated VAM after 28 days. Overall, this study showed that: (i) VAM fungi are a component of the soil environment in Hawkesbury Sandstone soils and mycorrhizal associations exist in a high proportion of the indigenous plant species, (ii) most of the propagules that can initiate V A M occur in the top 15 c m of soil, (iii) cutting soil blocks longitudinally into four and nine equal portions has no significant impact upon VAM fungi, but cutting blocks into 25 portions, temporarily can reduce the infective potential of VAM fungi to nil, and (iv) colonized root fragments and spores can be effective propagules initiating VAM after topsoil disturbance

Electronic Nose and GC-MS Analysis to Detect Mango Twig Tip Dieback in Mango (<i>Mangifera indica</i>) and Panama Disease (TR4) in Banana (<i>Musa acuminata</i>)

Volatile organic compounds (VOCs), as a biological element released from plants, have been correlated with disease status. Although analysis of VOCs using GC-MS is a routine procedure, it has limitations, including being time-consuming, laboratory-based, and requiring specialist training. Electronic nose devices (E-nose) provide a portable and rapid alternative. This is the first pilot study exploring three types of commercially available E-nose to assess how accurately they could detect mango twig tip dieback and Panama disease in bananas. The devices were initially trained and validated on known volatiles, then pure cultures of Pantoea sp., Staphylococcus sp., and Fusarium odoratissimum, and finally, on infected and healthy mango leaves and field-collected, infected banana pseudo-stems. The experiments were repeated three times with six replicates for each host-pathogen pair. The variation between healthy and infected host materials was evaluated using inbuilt data analysis methods, mainly by principal component analysis (PCA) and cross-validation. GC-MS analysis was conducted contemporaneously and identified an 80% similarity between healthy and infected plant material. The portable C 320 was 100% successful in discriminating known volatiles but had a low capability in differentiating healthy and infected plant substrates. The advanced devices (PEN 3/MSEM 160) successfully detected healthy and diseased samples with a high variance. The results suggest that E-noses are more sensitive and accurate in detecting changes of VOCs between healthy and infected plants compared to headspace GC-MS. The study was conducted in controlled laboratory conditions, as E-noses are highly sensitive to surrounding volatiles

The mitogenome of Phytophthora agathidicida: Evidence for a not so recent arrival of the "kauri killing" Phytophthora in New Zealand.

Phytophthora agathidicida is associated with a root rot that threatens the long-term survival of the iconic New Zealand kauri. Although it is widely assumed that this pathogen arrived in New Zealand post-1945, this hypothesis has yet to be formally tested. Here we describe evolutionary analyses aimed at evaluating this and two alternative hypotheses. As a basis for our analyses, we assembled complete mitochondrial genome sequences from 16 accessions representing the geographic range of P. agathidicida as well as those of five other members of Phytophthora clade 5. All 21 mitogenome sequences were very similar, differing little in size with all sharing the same gene content and arrangement. We first examined the temporal origins of genetic diversity using a pair of calibration schemes. Both resulted in similar age estimates; specifically, a mean age of 303.0-304.4 years and 95% HPDs of 206.9-414.6 years for the most recent common ancestor of the included isolates. We then used phylogenetic tree building and network analyses to investigate the geographic distribution of the genetic diversity. Four geographically distinct genetic groups were recognised within P. agathidicida. Taken together the inferred age and geographic distribution of the sampled mitogenome diversity suggests that this pathogen diversified following arrival in New Zealand several hundred to several thousand years ago. This conclusion is consistent with the emergence of kauri dieback disease being a consequence of recent changes in the relationship between the pathogen, host, and environment rather than a post-1945 introduction of the causal pathogen into New Zealand

A LAMP at the end of the tunnel: A rapid, field deployable assay for the kauri dieback pathogen, Phytophthora agathidicida.

The root rot causing oomycete, Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14-20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection of P. agathidicida that targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of other Phytophthora isolates and detected as little as 1 fg of total P. agathidicida DNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended soil bioassay and LAMP testing of DNA extracted from baits. In these comparisons, P. agathidicida was detected more frequently using the LAMP test. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the extended soil bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions about P. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid LAMP bioassay also has the potential to empower local communities to evaluate the pathogen status of local kauri stands, providing information for disease management and conservation initiatives

Cross-Cultural Leadership Enables Collaborative Approaches to Management of Kauri Dieback in Aotearoa New Zealand

In Aotearoa/New Zealand, the soilborne pathogen Phytophthora agathidicida threatens the survival of the iconic kauri, and the ecosystem it supports. In 2011, a surveillance project to identify areas of kauri dieback caused by Phytophthora agathidicida within the Wait&#257;kere Ranges Regional Park (WRRP) highlighted the potential impact of the pathogen. A repeat of the surveillance in 2015/16 identified that approximately a quarter of the kauri area within the Regional Park was infected or possibly infected, an increase from previous surveys. The surveillance program mapped 344 distinct kauri areas and showed that 33.4% of the total kauri areas were affected or potentially affected by kauri dieback and over half (58.3%) of the substantial kauri areas (above 5 ha in size) were showing symptoms of kauri dieback. Proximity analysis showed 71% of kauri dieback zones to be within 50 m of the track network. Spatial analysis showed significantly higher proportions of disease presence along the track network compared to randomly generated theoretical track networks. Results suggest that human interaction is assisting the transfer of Phytophthora agathidicida within the area. The surveillance helped trigger the declaration of a cultural ban (r&#257;hui) on recreational access. Te Kawerau &#257; Maki, the iwi of the area, placed a r&#257;hui over the kauri forest eco-system of the Wait&#257;kere Forest (Te Wao Nui o Tiriwa) in December 2017. The purpose of the r&#257;hui was to help prevent the anthropogenic spread of kauri dieback, to provide time for investment to be made into a degraded forest infrastructure and for research to be undertaken, and to help protect and support forest health (a concept encapsulated by the term mauri). Managing the spread and impact of the pathogen remains an urgent priority for this foundation species in the face of increasing pressures for recreational access. Complimentary quantitative and qualitative research programs into track utilization and ecologically sensitive design, collection of whakapapa seed from healthy and dying trees, and remedial phosphite treatments are part of the cross-cultural and community-enabled biosecurity initiatives to Kia Toitu He Kauri &ldquo;Keep Kauri Standing&rdquo;

Cross-Cultural Leadership Enables Collaborative Approaches to Management of Kauri Dieback in Aotearoa New Zealand

In Aotearoa/New Zealand, the soilborne pathogen Phytophthora agathidicida threatens the survival of the iconic kauri, and the ecosystem it supports. In 2011, a surveillance project to identify areas of kauri dieback caused by Phytophthora agathidicida within the Waitākere Ranges Regional Park (WRRP) highlighted the potential impact of the pathogen. A repeat of the surveillance in 2015/16 identified that approximately a quarter of the kauri area within the Regional Park was infected or possibly infected, an increase from previous surveys. The surveillance program mapped 344 distinct kauri areas and showed that 33.4% of the total kauri areas were affected or potentially affected by kauri dieback and over half (58.3%) of the substantial kauri areas (above 5 ha in size) were showing symptoms of kauri dieback. Proximity analysis showed 71% of kauri dieback zones to be within 50 m of the track network. Spatial analysis showed significantly higher proportions of disease presence along the track network compared to randomly generated theoretical track networks. Results suggest that human interaction is assisting the transfer of Phytophthora agathidicida within the area. The surveillance helped trigger the declaration of a cultural ban (rāhui) on recreational access. Te Kawerau ā Maki, the iwi of the area, placed a rāhui over the kauri forest eco-system of the Waitākere Forest (Te Wao Nui o Tiriwa) in December 2017. The purpose of the rāhui was to help prevent the anthropogenic spread of kauri dieback, to provide time for investment to be made into a degraded forest infrastructure and for research to be undertaken, and to help protect and support forest health (a concept encapsulated by the term mauri). Managing the spread and impact of the pathogen remains an urgent priority for this foundation species in the face of increasing pressures for recreational access. Complimentary quantitative and qualitative research programs into track utilization and ecologically sensitive design, collection of whakapapa seed from healthy and dying trees, and remedial phosphite treatments are part of the cross-cultural and community-enabled biosecurity initiatives to Kia Toitu He Kauri “Keep Kauri Standing”

Two new Nothophytophthora species from streams in Ireland and Northern Ireland: Nothophytophthora irlandica and N. lirii sp. nov

Slow growing oomycete isolates with morphological resemblance to Phytophthora were obtained from forest streams during routine monitoring for the EU quarantine forest pathogen Phytophthora ramorum in Ireland and Northern Ireland. Internal Transcribed Spacer (ITS) sequence analysis indicated that they belonged to two previously unknown species of Nothophytophthora, a recently erected sister genus of Phytophthora. Morphological and temperature-growth studies were carried out to characterise both new species. In addition, Bayesian and Maximum-Likelihood analyses of nuclear 5-loci and mitochondrial 3-loci datasets were performed to resolve the phylogenetic positions of the two new species. Both species were sterile, formed chlamydospores and partially caducous nonpapillate sporangia, and showed slower growth than any of the six known Nothophytophthora species. In all phylogenetic analyses both species formed distinct, strongly supported clades, closely related to N. chlamydospora and N. valdiviana from Chile. Based on their unique combination of morphological and physiological characters and their distinct phylogenetic positions the two new species are described as Nothophytophthora irlandica sp. nov. and N. lirii sp. nov. Their potential lifestyle and geographic origin are discussed