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), β-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.


Introduction
Macadamia (Macadamia integrifolia) is a tree nut crop that is cultivated for its highvalue kernel in tropical and subtropical regions in Australia, Asia, Africa, South America, and the U.S.A. Four species, M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii, are native to Australia [1]. Macadamia integrifolia and M. tetraphylla produce edible kernels, whereas M. ternifolia and M. jansenii produce small and inedible nuts that contain high levels of cyanogenic glycosides [2]. Several new diseases caused by fungal and oomyceteous pathogens have been reported on macadamia with the expansion of its production area [3][4][5].
Diseases of flowers and fruit result in significant yield losses and poor-quality kernels [6][7][8]. A mature macadamia tree may produce over 10,000 racemes (inflorescences) at peak anthesis, with each raceme typically having 100-300 flowers (Figure 1a) [9,10]. Flower diseases can result in poor pollination efficiency, with less than 1% of the flowers producing fruit, as well as a reduction in the potential of the flower to bear fruit. Macadamia racemes may be affected by fungal pathogens at different developmental stages. Macadamia inflorescences have four growth stages: small green buds on the rachis (stage 1); florets that turn from light green to white and are partially up to fully open, with stamens that pull away from the stigmas (stage 2); fully opened flowers with sepals that turn brown at peak anthesis (stage 3); sepals that drop and flowers with swollen fertilized embryos (stage 4) [11]. Most of the diseases that affect macadamia inflorescences are flower blights [12]. A diversity of fungi has been associated with flower blights of macadamia in Australia [13], including species of Botrytis [14], Cladosporium [15], Neopestalotiopsis, and Pestalotiopsis [8]. [13], including species of Botrytis [14], Cladosporium [15], Neopestalotiopsis, and Pestalotiopsis [8]. The incidence of macadamia dry flower disease caused by Pestalotiopsis macadamiae and Neopestalotiopsis macadamiae is on the increase in Australian macadamia plantations [8]. Dry flower disease is characterized by the necrotic blight of flowers ( Figure 1b). Akinsanmi et al. [16] suggested that multiple species of Pestalotiopsis and Neopestalotiopsis were responsible for dry flower epidemics in Australia.
Several species of Neopestalotiopsis are phytopathogens in tropical and subtropical regions, causing leaf spot, dry flower, fruit rot, fruit scab, and trunk diseases on a range of crops [5,8,[20][21][22][23][24][25][26]. Flower and leaf diseases on macadamia caused by Neopestalotiopsis spp. have been reported in Australia [5,8], Brazil [26], and China [25]. Many new pestalotioid species have been introduced in recent years [27][28][29][30][31][32][33]. Many unidentified isolates of Neopestalotiopsis were obtained from macadamia racemes with dry flower symptoms. However, there is little information about their identity and the diversity of fungi that cause dry flower disease on macadamia in Australia. The aim of this study was to identify the species of Neopestalotiopsis associated with the dry flowers of macadamia in Australia.

Sample Collection and Isolation
The isolates included in this study were collected from macadamia racemes with symptoms of dry flower disease. Samples were obtained from commercial macadamia orchards in Queensland (QLD) and New South Wales (NSW), Australia in 2019 and 2020. The samples were surface sterilized and incubated, as described by Akinsanmi et al. [8]. The incidence of macadamia dry flower disease caused by Pestalotiopsis macadamiae and Neopestalotiopsis macadamiae is on the increase in Australian macadamia plantations [8]. Dry flower disease is characterized by the necrotic blight of flowers ( Figure 1b). Akinsanmi et al. [16] suggested that multiple species of Pestalotiopsis and Neopestalotiopsis were responsible for dry flower epidemics in Australia.
Several species of Neopestalotiopsis are phytopathogens in tropical and subtropical regions, causing leaf spot, dry flower, fruit rot, fruit scab, and trunk diseases on a range of crops [5,8,[20][21][22][23][24][25][26]. Flower and leaf diseases on macadamia caused by Neopestalotiopsis spp. have been reported in Australia [5,8], Brazil [26], and China [25]. Many new pestalotioid species have been introduced in recent years [27][28][29][30][31][32][33]. Many unidentified isolates of Neopestalotiopsis were obtained from macadamia racemes with dry flower symptoms. However, there is little information about their identity and the diversity of fungi that cause dry flower disease on macadamia in Australia. The aim of this study was to identify the species of Neopestalotiopsis associated with the dry flowers of macadamia in Australia.

Sample Collection and Isolation
The isolates included in this study were collected from macadamia racemes with symptoms of dry flower disease. Samples were obtained from commercial macadamia orchards in Queensland (QLD) and New South Wales (NSW), Australia in 2019 and 2020. The samples were surface sterilized and incubated, as described by Akinsanmi et al. [8]. Monoconidial cultures of 13 isolates were established, as described by Akinsanmi et al. [34], and cryopreserved in a sterile 15% glycerol solution at −80 • C. Living cultures of the isolates were deposited in the Queensland Plant Pathology Herbarium (BRIP), Brisbane, Australia.

Cultural and Morphological Studies
Colony characteristics of cultures on 1 2 -potato dextrose agar (PDA; Difco Laboratories, Franklin Lakes, NJ, USA.) medium were recorded after 7 d incubation at 25 • C. Fungal morphology was recorded from colonies grown in the dark for 14 d at 25 • C on PDA as well as on autoclaved pine needles on water agar. Fungal structures were examined in lactic acid on slide mounts under a Leica DM5500B compound microscope (Wetzlar, Germany) with Nomarski differential interference contrast illumination, and images were taken with a Leica DFC 500 camera. Measurements of at least 30 conidia and other fungal structures were taken at 1000× magnification. Novel species were registered in MycoBank [35].

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from approx. 40 mg mycelium from colonies grown on PDA for 14 d. The mycelium was homogenized using TissueLyser (Qiagen, Chadstone, Australia) for 2 min at 30 Hz, and DNA was extracted using the BioSprint 96 DNA Plant Kit on a robotic platform (Qiagen, Chadstone, Australia). DNA concentration was determined with a BioDrop Duo spectrophotometer (BioDrop, Cambridge, England) and adjusted to 10 ng µL −1 . The DNA of each isolate served as the template for the PCR amplifications using the reactions and thermal cyclic conditions described by Prasannath et al. [5]. Briefly, each reaction was performed in a 25 µL reaction volume, with 1 µL each of 10 µM forward and reverse primers, PCR reaction mix, and 2 µL of DNA template. PCR amplification was performed in SuperCycler Thermal Cycler (Kyratec, Wembley, Australia) at 95 • C for 2 min, followed by 35 cycles at 95 • C for 30 s, 55 • C for 30 s, and at 72 • C for 1 min, with a final extension step at 72 • C for 5 min. Three loci, ITS, TUB, and TEF1α, were amplified and sequenced using the primer pairs ITS4/ITS5 [36], BT2A/BT2B [37], and EF1-526F/EF1-1567R [38], respectively. The quality of PCR amplicons was checked on 1% agarose gel electrophoresis stained with GelRed (Biotium, Melbourne, Australia) under UV light by Molecular Imager GelDoc (Bio-Rad Laboratories Inc., Gladesville, Australia). The targeted PCR products were purified and sequenced in both directions at Macrogen Inc. (Seoul, South Korea).

Phylogenetic Analyses
The DNA sequences were assembled in Geneious Prime v. 2021.0.3 (Biomatters Ltd., San Diego, CA, USA.), manually trimmed and aligned to produce consensus sequences for each locus. The consensus sequences generated in this study were deposited in GenBank ( Table 1). The sequences were compared against the NCBI GenBank nucleotide database using BLASTn to check the closest phylogenetic matches. The sequences of the ex-type isolates of the Neopestalotiopsis species were retrieved from GenBank (Table 1) and aligned with the sequences generated from our isolates using MAFFT v. 7.3.8.8 [39] in Geneious. Ambiguously aligned positions in each multiple alignment were excluded using Gblocks v. 0.91b [40]. The concatenated three-locus sequence dataset (ITS + TEF1α + TUB) of Neopestalotiopsis consisted of 63 taxa, with the outgroup taxon Pestalotiopsis diversiseta MFLUCC 12-0287 ( Table 1). The combined sequence data matrix was manually improved with BioEdit v. 7.2.5 [41] and gaps were treated as missing data. Phylogenetic trees were generated from Maximum Likelihood (ML), Bayesian Inference (BI), and Maximum Parsimony (MP) analyses. ML analysis was implemented using RAxML v. 8.2.11 [50] in Geneious. The search option was set to rapid bootstrapping, and the analysis was run using the GTR-GAMMAI evolution model with 1000 bootstrap iterations. BI analysis was conducted with MrBayes v. 3.2.1 [51] in Geneious to calculate posterior probabilities by the Markov Chain Monte Carlo (MCMC) method. The GTR-GAMMAI nucleotide substitution model was applied in BI analysis. Four MCMC chains were run simultaneously, starting from random trees for 1,000,000 generations. The temperature of the heated chain was set to 0.15 and trees were sampled every 200 generations until the average standard deviation of split frequencies reached 0.01 (stop value). Burn-in was set at 25%, after which the likelihood values were stationary. MP analysis was performed with PAUP v. 4.0b10 [52]. Trees were inferred using a heuristic search strategy, with 100 random stepwise addition and tree-bisectionreconnection (TBR) branch swapping. Max-trees were set to 5000 and bootstrap support values were evaluated for tree branches with 1000 replications [53]. Phylograms were visualized in FigTree v. 1.4.4 [54] and annotated in Adobe Illustrator 2021.
The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept and a pairwise homoplasy index (PHI) test were used to determine species boundaries [55]. The PHI test was performed using SplitsTree4 v. 4.17.1 [56] to determine the recombination level within phylogenetically closely related species. The concatenated three-locus dataset (ITS + TEF1α + TUB) was used for the analyses. PHI test results (Fw) >0.05 indicated no significant recombination within the dataset. The relationships between closely related taxa were visualized in split graphs with both the Log-Det transformation and splits decomposition options.

Phylogenetic Analyses
The concatenated sequence data matrix comprised 1367 base pairs (bp) (476 for ITS, 464 for TEF1α, and 427 for TUB), of which 935 bp were constant, 238 bp were parsimonyuninformative, and 174 bp were parsimony-informative. ML analysis yielded a best scoring tree, with a final ML optimization value of −6493.745 and the following model parameters
Culture characteristics: Colonies on PDA after 7 d at 25 °C reach 70 mm diam., with cream aerial mycelium, forming abundant pycnidia near the center after two weeks; reverse cream to buff.  N. javaensis, N. mesopotamica, and N. rosae [17].
Culture characteristics: Colonies on PDA after 7 d at 25 • C reach 80 mm diam., with white dense aerial mycelium, pycnidia abundant; reverse buff.
There was no evidence of significant genetic recombination (Fw > 0.05) between the novel species of Neopestalotiopsis and closely related species (Figure 8). The results confirmed that these taxa were significantly different from the existing species of Neopestalotiopsis. ngi 2021, 7, x FOR PEER REVIEW

Discussion
Five novel species, Neopestalotiopsis drenthii, N. maddoxii, N. olumi N. zakeelii, were discovered in isolates obtained from macadamia inflo flower disease and subsequently described. There was no evidence o recombination events between these species and their closest relatives Pestalotioid fungi (Pestalotiopsidaceae, Sordariomycetes) are s taxa, which are common pathogens on many crops [30,32,57]. Multi

Discussion
Five novel species, Neopestalotiopsis drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii, were discovered in isolates obtained from macadamia inflorescences with dry flower disease and subsequently described. There was no evidence of significant genetic recombination events between these species and their closest relatives.
There were 49 species names recognized in Neopestalotiopsis [58] prior to the five species described in this study. Neopestalotiopsis drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii formed well-supported monophyletic clades in the phylogenetic analysis. The topology of our phylogeny is similar to those generated in earlier studies [29,57].
Pestalotiopsis macadamiae and N. macadamiae were first reported as the causal fungi of dry flower disease of macadamia by Akinsanmi et al. [8]. These fungi are considered endemic to Australia and have likely co-evolved with macadamia. Pestalotiopsis macadamiae has been reported outside Australia on macadamia leaves in China [59]. The present study found a high diversity of Neopestalotiopsis spp. on macadamia racemes with dry flower symptoms. It is not known whether Neopestalotiopsis drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii are pathogens or saprobes. The role of these fungi in the pathogenicity of dry flower disease remains to be demonstrated by Koch's postulates. Living cultures of N. drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii are accessible in the BRIP culture collection for future research and comparative studies that may lead to the elucidation of their lifecycle and their role in dry flower disease.

Conclusions
Five fungal species, Neopestalotiopsis drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii, were described and illustrated. These fungi were isolated from inflorescences of macadamia with dry flower disease in Australia. The role that N. drenthii, N. maddoxii, N. olumideae, N. vheenae, and N. zakeelii play in dry flower disease of macadamia has yet to be determined. Hence, the pathogenicity of these novel species on macadamia racemes should be examined. Living cultures of the fungi are available for further study.