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.


Introduction
Macadamia species and hybrids (M. integrifolia × M. tetraphylla) are native to Australia and are now grown worldwide in tropical and subtropical regions for their nuts that have edible kernels [1]. The expansion of macadamia orchards into new regions has led to an increase in the number and severity of diseases caused by fungi and oomycetes [2][3][4]. Flower and fruit diseases reduce the nut set and can cause significant yield losses in commercial macadamia orchards [5][6][7]. A mature macadamia tree can produce more than 10,000 racemes (inflorescences) during the flowering period, with 100-300 flowers per raceme [8,9]. Fruit and flower diseases often cause poor pollination that can reduce the nut set by 99% [10]. Diverse fungal pathogens are associated with flower blights of macadamia including Botrytis cinerea [11], Cladosporium cladosporioides [12], Neopestalotiopsis macadamiae, and Pestalotiopsis macadamiae [7].
Under high humidity and moisture, B. cinerea causes grey mold (Botrytis blight) that covers infected macadamia flowers with mycelia ( Figure 1a) [11]. Index Fungorum accepted 71 Botrytis species (http://www.indexfungorum.org accessed on 17 September 2021), most of which are important pathogens of a wide range of host plants, including the grapevine, tomato, strawberry, bulbous crops, and cut flowers, causing devastating diseases during the preharvest and postharvest stages [13]. Among them, B. cinerea is one of the most important plant pathogens with wide-reaching economic and scientific impacts [14,15]. Many new species of Botrytis have been proposed [16] since Staats et al. [17] used molecular phylogenies to recognize Botrytis spp. The genus Cladosporium (Cladosporiaceae, Dothideomycetes) was introduced by Link [18] with C. herbarum (Pers.) Link as the type species. Cladosporium cladosporioides causes flower blight known as green mold (Cladosporium blight) that manifests as olive-grey-colored mycelial patches with abundant conidia on macadamia racemes that later become necrotic (Figure 1b) [12]. Cladosporium spp. include endophytes, pathogens, and saprobes, and have a worldwide distribution across a range of substrates [19][20][21][22][23]. Cladosporium spp. are well-known as plant pathogens [19,[24][25][26], and some can cause animal and human diseases [27][28][29]. Some pathogenic isolates of Cladosporium may have been wrongly classified as saprophytes, emphasizing the importance of the phylogenetic relationships for the identification of specialized lineages and cryptic species [24,28,30]. Some common species, C. cladosporioides, C. herbarum, and C. sphaerospermum, represent species complexes that await resolution as new isolates are collected from diverse ecosystems and geographical regions [19]. For example, C. polonicum and C. neapolitanum were described from within the C. cladosporioides species complex based on isolates recovered from galled flowers formed by midges on several species of Lamiaceae in Poland and Italy [31]. A phylogenetic analysis based on informative protein-coding genes is essential for the identification of species within Botrytis and Cladosporium genera [17,31]. Macadamia is a recently domesticated tree nut crop, with only B. cinerea and C. cladosporioides in their respective genera, reported as flower blight pathogens [11,12]. However, several unidentified isolates of Botrytis and Cladosporium were obtained from macadamia racemes with grey and green mold symptoms. Therefore, this study was aimed to determine the identity of the species of Botrytis and Cladosporium that are associated with flower diseases of macadamia in Australia.

Sample Collection and Isolation
The isolates included in this study were obtained from macadamia racemes with symptoms of grey and green mold diseases (Table 1). Samples were collected from commercial macadamia orchards in Queensland and New South Wales, Australia in 2019 and 2020. The specimens were surface sterilized and incubated, as described by Akinsanmi et al. [7]. Monoconidial cultures of the isolates were established, as described by Akinsanmi et al. [32], and preserved in a sterile 15% glycerol solution at −80 • C. Living cultures of the isolates were deposited in the Queensland Plant Pathology Herbarium (BRIP), Dutton Park, Australia.

Macro-and Micro-Morphological Studies
Colony characteristics of cultures on a 1 2 -potato dextrose agar (PDA; Difco Laboratories, Franklin Lakes, NJ, USA) medium were photographed after 14 d of incubation at 25 • C. The fungal morphology was recorded from colonies grown in the dark for 14 d at 25 • C on PDA. 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 captured 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 [33].

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from approx. 40 mg of 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). The DNA concentration was determined with a BioDrop Duo spectrophotometer (BioDrop, Cambridge, UK) and adjusted to 10 ng µL −1 . For Botrytis isolates, partial sequences of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene with primers G3PDHfor+ and G3PDHrev+ [17], DNA-dependent RNA polymerase subunit II (RPB2) gene with primers RPB2for+ and RPB2rev+ [17], and heat shock protein 60 (HSP60) gene with primers HSP60for+ and HSP60rev+ [17] were amplified. For Cladosporium isolates, amplification was carried out using primers ITS4 and ITS5 [34] for the internal transcribed spacer (ITS) region of rDNA, primers EF1-526F and EF1-1567R [35] for partial sequences of the translation elongation factor 1-alpha (TEF1α) gene, and primers ACT-512F and ACT-783R [36] for the actin (ACT) gene sequences. The DNA of each isolate served as the template for the PCR amplification. Each reaction was performed in a 25 µL reaction mixture containing 5 µL of 5 × reaction buffer (Bioline, Eveleigh, Australia), 1.5 µL of 25 mM MgCl 2 , 0.5 µL of 10 mM dNTPs, 1 µL each of 10 µM forward and reverse primers, 0.125 µL of MangoTaq DNA polymerase (5 U/µL; Bioline, Eveleigh, Australia), 13.875 µL of nuclease free water, and 2 µL of DNA template. Amplification was performed in a SuperCycler Thermal Cycler (Kyratec, Wembley, Australia) with initial denaturation at 95 • C for 2 min, followed by 35 cycles at 95 • C for 30 s, an annealing step at 55 • C for 30 s, and elongation at 72 • C for 1 min, with a final extension step at 72 • C for 5 min. 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, 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 (Tables 2 and 3). The sequences were compared against the NCBI GenBank nucleotide database using BLASTn to determine the closest phylogenetic relatives. The sequences of the reference isolates of the Botrytis (Table 2) and Cladosporium (Table 3) species were retrieved from GenBank and aligned with the sequences generated from our isolates using MAFFT v.7.3.8.8 [37] in Geneious. Ambiguously aligned positions in each multiple alignment were excluded using Gblocks v. 0.91b [38]. The concatenated three-locus sequence dataset (RPB2 + HSP60 + G3PDH) of Botrytis consisted of 42 taxa, with the outgroup taxon Sclerotinia sclerotiorum 484 ( Table 2). The combined ITS, TEF1α, and ACT sequences of isolates belonging to the C. cladosporioides species complex comprised 72 taxa, with the outgroup taxon C. herbarum CBS 121,621 ( Table 3). The combined sequence datasets were manually improved with BioEdit v. 7.2.5 [39], and gaps were treated as missing data. Phylogenetic trees were generated from Maximum Likelihood (ML), Bayesian Inference (BI), and Maximum Parsimony (MP) analyses. Table 2. Botrytis species and isolates used in the phylogenetic analysis with GenBank accession numbers.

Species Isolate
GenBank Accession Numbers 1

G3PDH HSP60 RPB2
Botrytis aclada  The ML analysis was implemented using RAxML v. 8.2.11 [40] in Geneious. The search option was set to rapid bootstrapping, and the analysis was run using the GTR + G + I substitution model with 1000 bootstrap iterations. The BI analysis was conducted with MrBayes v. 3.2.1 [41] in Geneious to calculate posterior probabilities by the Markov Chain Monte Carlo (MCMC) method. The GTR + G + I evolution model was applied in the 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 0.25, 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. The MP analysis was performed with PAUP v. 4.0b10 [42]. Trees were inferred using a heuristic search strategy with a 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 [43]. Phylograms were visualized in FigTree v. 1.4.4 [44] and annotated in Adobe Illustrator 2021.

Phylogenetic Analyses
The concatenated sequence data matrix of Botrytis comprised 2950 base pairs (bp) (1093 for RPB2, 971 for HSP60, and 886 for G3PDH), of which 2240 bp were constant, 296 bp were parsimony uninformative, and 414 bp were parsimony informative. The ML analysis yielded a best scoring tree, with a final ML optimization value of −11,930. The tree topology generated by the ML analysis was similar to that of the BI and MP analyses. The best scoring ML phylograms of Botrytis and Cladosporium are shown in Figures 2 and 3, respectively. ML bootstrap values, BI posterior probabilities, and MP bootstrap values (MLBS/BIPP/MPBS) are given at nodes of the phylogenetic trees (Figures 2 and 3). The phylogenetic tree inferred from the concatenated alignment resolved the four Botrytis isolates associated with the grey mold symptoms into an independent monophyletic clade with high support that represents a novel species within the Botrytis genus ( Figure 2). The phylogram inferred from the combined sequence data assigned four Cladosporium isolates associated with the green mold symptoms into three well-supported monophyletic clades that represent novel species within the Cladosporium genus ( Figure 3).   Maximum Likelihood tree topology of Cladosporium based on a combined multi-locus alignment (ITS + TEF1α + ACT). Cladosporium herbarum CBS 121621 was used as an outgroup taxon. Maximum Likelihood bootstrap support values (>50%), Bayesian Inference posterior probabilities (>90%), and Maximum Parsimony bootstrap proportions (>50%) are displayed at the nodes, respectively. Isolates of the newly described species are depicted in red. Description: Hyphae hyaline to pale brown, septate, 3-8 µm wide. Sclerotia single, sparse, dark grey to black, irregular to spherical, immersed, scattered, 0.2-2 mm diam. Conidiophores branched at top, erect, septate, subhyaline to pale brown, 1020-2050 × 10-20 µm. Conidiogenous cells swollen at the apex, 10-12 × 12-14 µm. Conidia in botryose clusters, elliptical to ovoid, unicellular, hyaline to pale brown, 9-11 × 6-7.5 µm.
Culture characteristics: Colonies on PDA 70 mm diam. after 14 d at 25 • C, flat, olivaceous, with sparse aerial mycelium, margins even and smooth; reverse black.

Discussion
Botrytis macadamiae, Cladosporium devikae, C. macadamiae, and C. proteacearum were isolated from macadamia inflorescences with grey and green mold symptoms and subsequently described. Each species formed a well-supported monophyletic clade in the phylogenetic analysis. The ITS region of the nuclear rDNA discriminates Botrytis from other genera in Sclerotiniaceae, although ITS is not useful for the delineation of the Botrytis species [45]. The three nuclear protein-coding genes, G3PDH, HSP60, and RPB2, have been used to characterize the Botrytis species [17]. To date, 40 species are phylogenetically recognized in Botrytis [16,46], including B. macadamiae. Whether B. macadamiae causes grey mold in macadamia has yet to be ascertained.
Grey mold is the most common disease caused by the Botrytis species affecting different plant organs, including flowers, fruits, leaves, and shoots [47]. Vegetables and small fruit crops such as the tomato, raspberry, grape, strawberry, blueberry, apple, and pear are among the most severely affected by these pathogens [47]. The genus Botrytis consisting of necrotrophic species has a very broad host range (e.g., B. cinerea and B. pseudocinerea) impacting more than 1400 different plant species [13]. On the contrary, other species have a narrow host range or are even host-specific, including B. fabae (broad bean) and B. calthae (marsh marigold) [48]. In some circumstances, multiple Botrytis species co-infect the same host plant; e.g., B. squamosa, B. allii, and B. aclada all cause significant economic risk to commercial onion production [15]. Interestingly, B. squamosa is family-specific and pathogenic on the onion, garlic, and leek (Allium spp.), while the closely related sister species are restricted to the lily (B. elliptica) and daylily (B. deweyae) [49]. Diversity among the Botrytis species is shown by cultural characteristics, virulence, and host specificity. However, the unique feature among all grey mold fungi is their necrotrophic lifestyle in which they kill host cells via the secretion of effector proteins to induce cell death, obtain nutrients, and subsequently colonize dead plant tissue [49,50].
The Cladosporium species are known as common and abundant fungi in indoor and outdoor environments. The Cladosporium species are also economically important spoilage organisms of grains, fruits, and refrigerated meat [51][52][53]. Several Cladosporium species are pathogenic to a wide range of hosts [30]. Most Cladosporium species are saprobic, but some have also been reported as endophytes, phylloplane fungi, and hyperparasites on other fungi [54][55][56]. Certain species show the ability to produce compounds of medical interest or are relevant as potential biocontrol agents for plant diseases [57,58]. Some species are pathogens to various crops and can cause economically important diseases, while others have only endemic importance [59]. These fungi can cause diseases of plants, often with different names, depending on the infected plants and the type of symptoms. Pathogenic species of Cladosporium are known to cause leaf mold of the tomato [60] and scab disease on leaves of the cucumber, the strawberry, and tea [61][62][63]. Cladosporium cladosporioides has been reported as a pathogen of scab in papaya [64], sooty mold in the persimmon [65], blossom blight in the strawberry [66], and leaf spot in the tomato [67].
Three major species complexes are recognized within the genus Cladosporium, viz. the C. cladosporioides, C. herbarum, and C. sphaerospermum species complexes [30]. The species identification and delineation in Cladosporium require a multi-locus DNA sequence analysis of the ITS region of rDNA gene, partial ACT, and TEF1α gene sequences [30]. The molecular approach combined with morphological features allowed the recognition of more than 230 species within the genus Cladosporium [68]. Our phylogenetic analysis using these three loci placed C. devikae, C. macadamiae, and C. proteacearum in the C. cladosporioides species complex. These species were well-delineated from other species in the C. cladosporioides species complex.
The proper identification of species is essential for all biological studies. The present study found a high diversity of Cladosporium spp. on macadamia racemes with green mold symptoms. Future studies will determine whether B. macadamiae, C. devikae, C. macadamiae, and C. proteacearum are pathogens or saprobes on macadamia inflorescences. Living cultures of B. macadamiae, C. devikae, C. macadamiae, and C. proteacearum are preserved and accessible in BRIP as cryopreserved cultures for future research and study.

Conclusions
Botrytis macadamiae, Cladosporium devikae, C. macadamiae, and C. proteacearum were described and illustrated. These fungi were isolated from inflorescences of macadamia with grey and green mold symptoms in Australia. The pathogenicity of these novel species on macadamia racemes has yet to be examined. Cryopreserved isolates of these fungi are available in BRIP for future research. Data Availability Statement: All sequence data are available in NCBI GenBank (www.ncbi.nlm.nih. gov) following the accession numbers in the manuscript.