Effect of Sampling Time, Quantification Method, and Tree Sample Pooling on Phytophthora cinnamomi Root Quantities in South African Avocado Orchards.

Phytophthora cinnamomi is a destructive soilborne pathogen causing Phytophthora root rot on avocados worldwide. Little is known about the effect of root sampling time, root quantification method (quantitative real-time PCR [qPCR] versus baiting), and tree sample pooling strategies on the quantification of the pathogen in roots in avocado orchard trees. This was investigated in six avocado orchards in two climatically different production regions (Mooketsi and Letaba) in the Limpopo Province, South Africa, over a 2-year period. Two different tree sample pooling strategies, consisting of either a four-pooled group (four groups each containing five pooled trees) or a single-pooled group (20 trees pooled) per 1 ha, were both shown to be suitable for quantifying P. cinnamomi in tree roots using qPCR or root baiting. P. cinnamomi root quantities from the two tree sample pooling strategies were significantly correlated for both quantification methods. Both quantification methods were suitable for quantifying the pathogen in roots, although qPCR was superior to root baiting at identifying significant differences in P. cinnamomi quantities among root sampling time points. The effect of sampling time was dependent on the investigated year. In 2017, root quantities, which were only evaluated using qPCR, did not reveal a consistent trend of a specific sampling time yielding the highest root quantities for most of the orchards. However, five of the orchards in 2018, based on the qPCR analyses, contained significantly higher P. cinnamomi root quantities in May (late autumn) than in March (early autumn), August (late winter), and October/November (late spring). In 2018, P. cinnamomi root DNA quantities were significantly positively correlated with the number of soil temperature hours at 20 to 24 and 20 to 29°C 2 months preceding the root sampling dates and negatively correlated with the number of hours at 15 to 19°C 2 months preceding root sampling. Our study has identified P. cinnamomi root quantification methods and tree sample pooling strategies, which will be useful for understanding the biology of the pathogen and when disease management strategies should be in place.

Evaluation of Fungicide Soil Drench Treatments to Manage Black Root Rot Disease of Avocado.

Four common fungicidal products were evaluated for their effect on symptoms caused by two nectriaceous black root rot fungi, Calonectria ilicicola and Dactylonectria macrodidyma, when applied as pot drenches to avocado (Persea americana) seedlings in the greenhouse. Applications of fludioxonil, thiophanate-methyl + etridiazole, prochloraz, and prochloraz MnCl at 2 and 4 weeks after inoculation with C. ilicicola significantly reduced root necrosis and improved root and aboveground plant biomass compared with water-treated controls. Fludioxonil reduced necrosis by 60% and had a significantly lower frequency of reisolation of C. ilicicola than the other three fungicide treatments. D. macrodidyma inoculation caused less severe symptoms in seedlings than C. ilicicola despite the longer duration of the trial. Pot drenches with fludioxonil, thiophanate-methyl + etridiazole, and prochloraz MnCl, but not prochloraz alone, significantly reduced root necrosis caused by D. macrodidyma. Prochloraz MnCl was the only fungicide treatment to increase root and plant biomass compared with water-treated controls. Both fludioxonil and prochloraz MnCl reduced the frequency of reisolation of D. macrodidyma from necrotic roots by about 50% compared with the other fungicides or water controls. The results indicated that drenches with these fungicides may suppress existing low to moderate black root rot infection, allowing new root growth and improved establishment in the orchard. Fungicide drenching must not replace best-practice disease management strategies in nurseries but may be a useful tool in crisis situations.

Diversity and Pathogenicity of Botryosphaeriaceae Associated with Macadamia Branch Dieback in Australia.

Botryosphaeria branch dieback is a serious disease of macadamia in Australia, but its etiology has not been clearly defined, which limits effective disease control. Therefore, this study examined whether the causal agents of branch dieback in commercial macadamia orchards in five agroecological regions in Australia are similar in prevalence and aggressiveness. The identity of the causal agents was determined using conventional culturing techniques and DNA sequencing that targets the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1α), ß-tubulin (tub2), and DNA-directed RNA polymerase II second largest subunit (rpb2) gene loci. The pathogenic variation of the isolates, relative to the source (region and host plant part), was examined using in vivo and in planta assays. Lasiodiplodia and Neofusicoccum were the dominant fungal genera obtained from surveys of 59 macadamia orchards across the agroecological regions. Phylogenetic analysis of 52 representative isolates identified four putative novel Lasiodiplodia clades, with three other Lasiodiplodia spp. (Lasiodiplodia iraniensis, L. pseudotheobromae, and L. theobromae) and three Neofusicoccum spp. (Neofusicoccum luteum, N. mangroviorum, and N. parvum) from macadamia. L. pseudotheobromae that constituted 40% of the isolates from symptomatic tissues was the most prevalent in all the regions. Both the in vivo and in planta pathogenicity assays revealed that all isolates of the Botryosphaeriaceae, except N. mangroviorum, were pathogenic to macadamia. L. theobromae, N. luteum, and L. iraniensis were the most aggressive species causing dieback symptoms in macadamia.

Development of Three Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Calonectria ilicicola, Dactylonectria macrodidyma, and the Dactylonectria Genus in Avocado Roots.

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from ß-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.

Effects of Silicon Amendment on Soilborne and Fruit Diseases of Avocado.

The effects of silicon (Si) amendment have been studied in several plant/pathogen interactions; however, studies in horticultural tree crops are limited. Effects of amendment with soluble potassium silicate (AgSil®32, approximately 30% available Si), or milled cement building board by-products (Mineral Mulch (MM) or Mineral Dust (MD), containing 5% available Si) were investigated in field and greenhouse trials with avocado. Orchard soil drench applications with potassium silicate improved yield and quality of fruit, but visual health of trees declining from Phytophthora root rot (PRR) was not affected. Orchard spray or trunk injection applications with potassium silicate were ineffective. Amendment of potting mix with MM and MD reduced root necrosis of avocado seedlings after inoculation with Calonectria ilicicola, an aggressive soilborne pathogen causing black root rot. Application of MM to mature orchard trees declining with PRR had a beneficial effect on visual tree health, and Si accumulation in leaves and fruit peel, after only 10 months. Products that deliver available Si consistently for uptake are likely to be most successful in perennial tree crops.

Pathogenicity of Nectriaceous Fungi on Avocado in Australia.

Black root rot is a severe disease of young avocado trees in Australia causing black necrotic roots, tree stunting, and leaf drop prior to tree death. Nectriaceous fungi (Nectriaceae, Hypocreales), are commonly isolated from symptomatic roots. This research tested the pathogenicity of 19 isolates from Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, and Ilyonectria, spp. collected from young avocado trees and other hosts. Glasshouse pathogenicity tests with 'Reed' avocado (Persea americana) seedlings confirmed that Calonectria ilicicola is a severe pathogen of avocado, causing stunting, wilting, and seedling death within 5 weeks of inoculation. Isolates of C. ilicicola from peanut, papaya, and custard apple were also shown to be aggressive pathogens of avocado, demonstrating a broad host range. An isolate of a Calonectria sp. from blueberry and avocado isolates of Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, and D. anthuriicola caused significant root rot but not stunting within 5 to 9 weeks of inoculation. An isolate of an Ilyonectria sp. from grapevine closely related to Ilyonectria liriodendri, and avocado isolates of Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii, and an Ilyonectria sp. were not pathogenic to avocado.

Effects of silicon treatment and inoculation with Fusarium oxysporum f. sp. vasinfectum on cellular defences in root tissues of two cotton cultivars.

BACKGROUND AND AIMS: Silicon has been shown to enhance the resistance of plants to fungal and bacterial pathogens. Here, the effect of potassium silicate was assessed on two cotton (Gossypium hirsutum) cultivars subsequently inoculated with Fusarium oxysporum f. sp. vasinfectum (Fov). Sicot 189 is moderately resistant whilst Sicot F-1 is the second most resistant commercial cultivar presently available in Australia. METHODS: Transmission and light microscopy were used to compare cellular modifications in root cells after these different treatments. The accumulation of phenolic compounds and lignin was measured. KEY RESULTS: Cellular alterations including the deposition of electron-dense material, degradation of fungal hyphae and occlusion of endodermal cells were more rapidly induced and more intense in endodermal and vascular regions of Sicot F-1 plants supplied with potassium silicate followed by inoculation with Fov than in similarly treated Sicot 189 plants or in silicate-treated plants of either cultivar not inoculated with Fov. Significantly more phenolic compounds were present at 7 d post-infection (dpi) in root extracts of Sicot F-1 plants treated with potassium silicate followed by inoculation with Fov compared with plants from all other treatments. The lignin concentration at 3 dpi in root material from Sicot F-1 treated with potassium silicate and inoculated with Fov was significantly higher than that from water-treated and inoculated plants. CONCLUSIONS: This study demonstrates that silicon treatment can affect cellular defence responses in cotton roots subsequently inoculated with Fov, particularly in Sicot F-1, a cultivar with greater inherent resistance to this pathogen. This suggests that silicon may interact with or initiate defence pathways faster in this cultivar than in the less resistant cultivar.

An inordinate fondness for Fusarium: phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts.

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naïve natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Clade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene ∼21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization.