Fungal composition, quantification of mycotoxins, and enzyme activity in processed Solanum tuberosum Linn (potato) products stored at different relative humidity.

AIM: Postharvest loss of potatoes at the peak of harvest is of global concern. This study aimed to determine the quality of stored processed potato products based on fungal composition, mycotoxin contamination, and fungal enzyme activity. MATERIALS AND METHODS: Potato products from three cultivars (Caruso, Marabel, and Nicola) were grouped as peeled or unpeeled, oven- or sun-dried, and all samples were in flour form. Samples were incubated separately for 6 weeks at 25%, 74%, and 87% relative humidities (RH) at 25°C. The pH, moisture content (MC), visible deterioration, mycotoxin, fungal identity by DNA sequencing, and enzyme activity were determined. RESULTS: Results of grouped products (based on variety, drying, and peeling method) revealed that MC increased in the oven-dried samples and the pH value reduced after incubation. About 26% of the products at 87% RH showed visible deterioration, low amounts of fumonisin were detected in fermented potato product and nine fungal genera were identified across the three RH levels. Enzyme activities by Aspergillus niger, Fusarium circinatum, and Rhizopus stolonifer isolates were confirmed. CONCLUSION: RH influenced deterioration and fungal activities in some stored processed potato products. Low levels of fumonisin were detected.

Evaluation of In-Field Inoculation Methods for Characterization of Macadamia Germplasm to Husk Spot Incidence and Severity.

Husk spot, a fungal disease of macadamia pericarps (Pseudocercospora macadamiae), induces premature abscission in several major commercial cultivars. Breeding for resistance to husk spot is a priority of the Australian macadamia industry. Due to the large tree size of macadamia and high numbers of progeny in breeding populations, inoculating for resistance screening is laborious and time consuming. Previously utilized methods included direct applications of P. macadamiae suspensions and the hanging of bags of diseased husks above developing fruit in tree canopies. In this study, both methods were modified to allow for efficient application in large-scale breeding populations, and their efficacy was evaluated. Two quantities of diseased husk per bag, 'large' (75 g) and 'small' (30 g), and two concentrations of sprayed P. macadamiae suspensions, 'stock' (5 × 105 propagules/ml) and 'dilute' (2.5 × 105 propagules/ml), were tested across two fruiting seasons. Treatments were compared against a control (sterile water) in commercial cultivars A38 and A4. Husk spot incidence and severity produced by small bags were significantly affected by season. A significant season effect was less common for other treatments. All four treatments infected over 50% of target fruit in each season, but the highest husk spot incidence across both seasons (≥85%) was produced from large bags. Overall, the large bags were the most reliable method for infection of target fruit. Results also demonstrate the importance of considering the effect of season when selecting husk spot inoculation methods.

Sources, Detection, and Inoculum Quantification of Flower Blight Pathogens in Macadamia.

Dry flower disease caused by Pestalotiopsis/Neopestalotiopsis spp., green mold caused by Cladosporium spp., and gray mold caused by Botrytis spp., collectively known as flower blight cause significant yield losses in macadamia. Potential sources of inoculum of the various pathogens in macadamia tree canopy were examined using pathogenicity tests and a multiplex quantitative PCR (qPCR) assay developed in this study. The qPCR assay detected and quantified the relative abundance of the inoculum of flower blight pathogens. The assay revealed that remnant racemes contributed a high amount of inoculum of all the three groups of flower blight pathogens, while the yellow halo leaf spot contributed only Pestalotiopsis/Neopestalotiopsis species. The amount of conidia per gram of remnant racemes ranged from 7 × 103 to 2 × 104 for dry flower disease, 3 × 103 to 1 × 104 for green mold, and 5 to 8 × 103 for gray mold pathogens. Conidia of Pestalotiopsis/Neopestalotiopsis species quantified from leaf spots varied from 1 × 102 to 1 × 103 per cm2. Pathogenicity tests performed on developing racemes under field conditions, using conidial suspensions from both sources of inoculum (remnant racemes and yellow halo leaf spot), resulted in severe flower bight symptoms. Disease severity was not significantly different (P > 0.05) when remnant racemes were incubated directly with the developing racemes compared with inoculation with conidial suspension from the material. This suggests that racemes from preceding seasons that remain in the tree canopy carryover inoculum between seasons and should be removed as a control option for flower blights in macadamia orchards.

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.

Influence of climatic factors on dry flower, grey and green mould diseases of macadamia flowers in Australia.

AIMS: Flower blights (grey mould, green mould and dry flower) are important diseases of macadamia. Lack of information on pathogen biology and disease epidemiology in macadamia has hampered control options. Effects of climatic variables including temperature, relative humidity (RH) and vapour pressure deficit (VPD) on the abundance, germination and growth of conidia of four fungal pathogens that cause various flower blights in macadamia were studied. METHODS AND RESULTS: Mycelial growth, sporulation, conidial germination and germ tube growth for five isolates each of Botrytis cinerea, Cladosporium cladosporioides, Pestalotiopsis macadamiae and Neopestalotiopsis macadamiae, at eight temperatures, seven RH and the corresponding VPD regimes were determined in vitro. The optimal climatic range of each of the four pathogens was validated during macadamia flowering periods in the 2019 and 2020 seasons by conidia detected and quantified using quantitative PCR. Several growth models were fitted to the data with high significance; predicted optima from these models ranged from 0.9 to 1.1 kPa VPD for P. macadamiae and N. macadamiae and <0.6 kPa VPD for B. cinerea and C. cladosporioides. CONCLUSIONS: This study showed that VPD, as a determinant of the fecundity and growth of the four fungal pathogens, was predictive of flower blight incidence in macadamia. The importance of temperature, RH and, thus, VPD for defining the conditions for infection and flower blight epidemics was established. SIGNIFICANCE AND IMPACT OF THE STUDY: This information provides a firm basis for the development of prediction tools for flower blights in macadamia.

Characterisation of novel endogenous geminiviral elements in macadamia.

BACKGROUND: The presence of geminivirus sequences in a preliminary analysis of sRNA sequences from the leaves of macadamia trees with abnormal vertical growth (AVG) syndrome was investigated. RESULTS: A locus of endogenous geminiviral elements (EGE) in the macadamia genome was analysed, and the sequences revealed a high level of deletions and/or partial integrations, thus rendering the EGE transcriptionally inactive. The replication defective EGE in the macadamia genome indicates its inability to be the source of new viral infections and thus cause AVG or any other disease in macadamia. The EGE sequences were detected in two edible Macadamia species that constitute commercial cultivars and the wild germplasm of edible and inedible species of Macadamia. This strongly suggests that the integration preceded speciation of the genus Macadamia. A draft genome of a locus of EGE in Macadamia was developed. The findings of this study provide evidence to suggest the endogenization of the geminiviral sequences in the macadamia genome and the ancestral relationship of EGE with Macadamia in the Proteaceae family. Random mutations accumulating in the EGE inform that the sequence is evolving. CONCLUSIONS: The EGE in Macadamia is inactive and thus not a direct cause of any diseases or syndromes including AVG in macadamia. The insertion of the EGE in the macadamia genome preceded speciation of the genus Macadamia.

Novel Botrytis and Cladosporium Species Associated with Flower Diseases of Macadamia in Australia.

Macadamia (Macadamia integrifolia) is endemic to eastern Australia and produces an edible nut that is widely cultivated in commercial orchards globally. A survey of fungi associated with the grey and green mold symptoms of macadamia flowers found mostly species of Botrytis (Sclerotiniaceae, Leotiomycetes) and Cladosporium (Cladosporiaceae, Dothideomycetes). These isolates included B. cinerea, C. cladosporioides, and unidentified isolates. Amongst the unidentified isolates, one novel species of Botrytis and three novel species of Cladosporium were delimited and characterized by molecular phylogenetic analyses. The new species are Botrytis macadamiae, Cladosporium devikae, C. macadamiae, and C. proteacearum.

Neopestalotiopsis Species Associated with Flower Diseases of Macadamia integrifolia in Australia.

Macadamia (Macadamia integrifolia) is native to eastern Australia and produces an edible nut that is extensively cultivated in commercial orchards in several countries. Little is known about the diversity of fungi associated with diseases of macadamia inflorescences. A survey of fungi associated with the dry flower disease of macadamia detected several isolates of Neopestalotiopsis (Pestalotiopsidaceae, Sordariomycetes). Five new species of Neopestalotiopsis were identified based on molecular phylogenetic analyses of concatenated gene sequences of the internal transcribed spacer (ITS), ß-tubulin (TUB), and the translation elongation factor 1-alpha (TEF1α). The new species are named Neopestalotiopsis drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii, and are described by molecular, morphological, and cultural characteristics. The ecology of the isolates and their pathogenic, saprophytic, or commensal ability were not determined.

Discovery of Single Nucleotide Polymorphisms for Resistance to Abnormal Vertical Growth in Macadamia.

Abnormal vertical growth (AVG) syndrome is a serious threat to the Australian macadamia industry as it decreases the yield of nuts by as much as 70% per annum. A lack of information on the cause of AVG has hindered the development of an effective disease management strategy. Discovery of genetic markers associated with disease resistance can be used as tool for rapid selection of elite cultivars, hence helps in efficient disease management. Differences in field susceptibility of macadamia cultivars provide an opportunity for discovery of genetic markers that are associated with host resistance. REML mixed model analysis was performed to estimate the AVG rating of 51 cultivars from multiple origins using phenotypic data from 359 trees planted in four sites. Most of the Hawaiian cultivars were found as susceptible, while selections from the Australian macadamia industry breeding program were predominantly resistant. All the cultivars were genotyped for 13,221 DArTseq-based single nucleotide polymorphism (SNP) markers. A bulked sample analysis was performed using 20 genotypes each at the extremes of AVG phenotypic ratings. Ten SNP markers were predicted to be associated with AVG resistance and two arbitrarily selected SNP markers were validated using PCR and Sanger sequencing. Our findings suggest that AVG resistance in the commercial cultivars may be derived from the genomic introgression of Macadamia tetraphylla through interspecific hybridization. The results may support marker-assisted selection for macadamia germplasm with AVG resistance.

Draft Genome of the Macadamia Husk Spot Pathogen, Pseudocercospora macadamiae.

Pseudocercospora macadamiae causes husk spot in macadamia in Australia. Lack of genomic resources for this pathogen has restricted acquiring knowledge on the mechanism of disease development, spread, and its role in fruit abscission. To address this gap, we sequenced the genome of P. macadamiae. The sequence was de novo assembled into a draft genome of 40 Mb, which is comparable to closely related species in the family Mycosphaerellaceae. The draft genome comprises 212 scaffolds, of which 99 scaffolds are over 50 kb. The genome has a 49% GC content and is predicted to contain 15,430 protein-coding genes. This draft genome sequence is the first for P. macadamiae and represents a valuable resource for understanding genome evolution and plant disease resistance.

Spatiotemporal Spread of Abnormal Vertical Growth of Macadamia in Australia Informs Epidemiology.

Australian macadamia production is threatened by a disorder known as abnormal vertical growth (AVG), for which the etiology is unknown. AVG is characterized by vigorous upright growth and reduced lateral branching, flowering, and nut set that results in over 70% yield loss annually. Six commercial macadamia orchards were surveyed in 2012 and again in 2018 to examine spatiotemporal dynamics of the epidemic. Data were subjected to point-pattern and geostatistical analyses. AVG incidence in all orchards showed a better fit to the beta-binomial distribution than the binomial distribution. AVG incidence in the different orchards varied between 5 and 47% in 2012, and 13 and 55% in 2018 and the rate of spread was slow, averaging at about 2% increase in disease incidence per annum. Spatial patterns of AVG were highly aggregated on both survey years and spread was mainly between neighboring trees in a row or trees that were opposite to each other in different rows. Semivariograms showed large range values (approximately 15 to 120), indicating aggregation of AVG-affected trees beyond quadrat levels. Furthermore, clusters of disease were mainly at the edge of the orchard on the first survey date and the disease progressed toward the center of the orchard over time. It is concluded that AVG is caused by an infectious agent, and based on patterns of spread, we hypothesize that spread is facilitated by root grafting or root-to-root contact. Furthermore, a vascular-limited pathogen could be involved that modulates plant hormone production.

Dry Flower Disease of Macadamia in Australia Caused by Neopestalotiopsis macadamiae sp. nov. and Pestalotiopsis macadamiae sp. nov.

Incidence of dry flower disease of macadamia (Macadamia integrifolia), expressed as blight of the flowers and necrosis and dieback of the rachis, is increasing in Australia. In the 2012-13 production season, incidence of dry flower disease resulted in 10 to 30% yield loss in the affected orchards. Etiology of the disease has not been established. This study was established to characterize the disease and identify the causal pathogen. A survey of the major macadamia-producing regions in Australia revealed dry flower disease symptoms regardless of cultivar or location at all stages of raceme development. Based on colony and conidial morphology, the majority (41%) of fungal isolates obtained from tissue samples were identified as Pestalotiopsis and Neopestalotiopsis spp. The phylogeny of the combined partial sequence of the internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α gene loci segregated the isolates into two well-supported clades, independent of location or part of the inflorescence affected. Further morphological examination supported the establishment of two new species, which are formally described as Neopestalotiopsis macadamiae sp. nov. and Pestalotiopsis macadamiae sp. nov. Using spore suspensions of isolates of both species, Koch's postulates were fulfilled on three macadamia cultivars at all stages of raceme development. To our knowledge, this is the first report of species of Neopestalotiopsis and Pestalotiopsis as causal agents of inflorescence disease in macadamia.

Passage through alternative hosts changes the fitness of Fusarium graminearum and Fusarium pseudograminearum.

Species of the necrotrophic fungal pathogen Fusarium that cause head blight and crown rot of cereals including wheat also infect a number of alternative host plants. This raises the prospect of more damaging pathogen strains originating and persisting as highly successful saprophytes on hosts other than wheat. The immediate impact on pathogenic (aggressiveness) and saprophytic (growth rate and fecundity) behaviour of six isolates with low, moderate or high initial aggressiveness was examined in two species of Fusarium after their passage through 10 alternative plant hosts. One passage through alternative hosts significantly reduced the pathogenic fitness of most isolates, but this change was not associated with a concomitant change in their overall saprophytic behaviour. The overall weak association between aggressiveness, fecundity and growth rate both before and after passage through the alternative hosts indicate that pathogenic and saprophytic fitness traits may be independently controlled in both Fusarium species. Thus, there was no trade-off between pathogenic and saprophytic fitness in these necrotrophic plant pathogens.