花生晚叶斑病病原个体尾孢霉的表型变异特征、形态、遗传和代谢产物-动脉网

Characterizing phenotype variants of Cercosporidium personatum, causal agent of peanut late leaf spot disease, their morphology, genetics and metabolites

Nature 等信源发布 2025-01-09 16:05

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Abstract

摘要

Cercosporidium personatum

人尾孢霉

(CP) causes peanut late leaf spot (LLS) disease with 70% yield losses unless controlled by fungicides. CP grows slowly in culture, exhibiting variable phenotypes. To explain those variations, we analyzed the morphology, genomes, transcriptomes and chemical composition of three morphotypes, herein called RED, TAN, and BROWN.

（CP）引起花生晚叶斑病（LLS），除非用杀菌剂控制，否则产量损失70%。CP在培养物中生长缓慢，表现出可变的表型。为了解释这些变异，我们分析了三种形态类型（此处称为红色，棕褐色和棕色）的形态，基因组，转录组和化学组成。

We characterized, for the first time in CP, anthraquinone (AQ) precursors of dothistromin (DOT), including averantin, averufin, norsolorinic acid, versicolorin B, versicolorin A, nidurufin and averufanin. BROWN had the highest AQ and melanin (15 mg/g DW) contents. RED had the highest ergosterol (855 µM FW) and chitin (beta-glucans, 4% DW) contents.

我们首次在CP中表征了dothistromin（DOT）的蒽醌（AQ）前体，包括阿维菌素，阿维鲁芬，去甲索酸，versicolorin B，versicolorin A，nidurufin和averufanin。棕色的AQ和黑色素含量最高（15 mg/g DW）。红色的麦角固醇（855µM FW）和几丁质（β-葡聚糖，4%DW）含量最高。

RED and TAN had higher resistance to xenobiotics (.

红色和棕色对异生素具有较高的抗性（。

≤ 1.0E-3), including chlorothalonil, tebuconazole and caffeine, compared to CP NRRL 64,463. In RED, TAN, and BROWN, rates of single nucleotide polymorphisms (SNP) (1.4–1.7 nt/kb) and amino acid changes (3k-4k) were higher than in NRRL 64,463. Differential gene expression (

≤1.0E-3），包括百菌清，戊唑醇和咖啡因，与CP NRRL 64463相比。在红色，棕褐色和棕色中，单核苷酸多态性（SNP）（1.4–1.7 nt/kb）和氨基酸变化（3k-4k）的发生率高于NRRL 64463。差异基因表达(

≤ 1.0E-5) was observed in 47 pathogenicity/virulence genes, 41 carbohydrate-active enzymes (CAZymes), and 23 pigment/mycotoxin biosynthesis genes. We describe the MAT1 locus, and a method to evaluate CP-xenobiotic resistance in 5 days. Chemical profiles indicate each CP morphotype could trigger different immune response in plants, probably hindering development of durable LLS resistance..

在47个致病性/毒力基因，41个碳水化合物活性酶（CAZymes）和23个色素/真菌毒素生物合成基因中观察到≤1.0E-5）。我们描述了MAT1基因座，以及在5天内评估CP异生素抗性的方法。化学特征表明，每种CP形态类型都可能在植物中引发不同的免疫反应，可能阻碍持久LLS抗性的发展。。

Introduction

导言

Slow growth and peculiar morphologies in culture have hindered the study of the fungus

生长缓慢和培养中的特殊形态阻碍了真菌的研究

Cercosporidium personatum

人尾孢霉

Berk. & Curtis (CP) (Syn.

伯克。柯蒂斯（CP）（儿子。

Nothopassalora personata

个人不可侵犯

(Berk. & Curtis) U. Braun, C. Nakash., Videira & Crous), causal agent of late leafspot (LLS) in peanut (

（伯克和柯蒂斯）U.Braun，C.Nakash。，Videira＆Crous），花生晚叶斑病（LLS）的致病因子(

Arachis hypogaea

花生

L.)

五十、）

. LLS together with early leafspot disease caused by

.LLS以及由

Cercospora arachidicola

花生煤油

Hori can result in yield losses of up to 70% if not controlled

如果不加以控制，Hori可能导致高达70%的产量损失

. Management of these diseases costs $40 million a year to peanut growers in the State of Georgia, U.S.A., who despite frequent fungicide applications experience losses of $12 million to leafspot

美国佐治亚州的花生种植者每年要花费4000万美元来管理这些疾病，尽管他们经常使用杀菌剂，但他们的叶斑病损失了1200万美元

. CP can be considered hemibiotroph/biotroph, as its close relative

CP可以被认为是半生物营养体/生物营养体，因为它是近亲

Cladosporium fulvum

黄枝孢菌

At present, effective control of peanut leaf spot disease in peanut is normally treated with multiple fungicide combinations that include chlorothalonil (Bravo), prothioconazole + tebuconazole (Provost), pydiflumetofen (Miravis), and azoxystrobin (Elatus), Virginia Tech, Nov 2020. In the early 1990s, chlorothalonil was used at rates of 1.2 kg ai/ha to control late leaf spot, though the dose could be lowered to 0.42 kg ai/ha when combined with cyproconazole.

目前，花生叶斑病的有效防治通常采用多种杀菌剂组合，包括百菌清（Bravo），丙硫唑 + 戊唑醇（Provost），pydiflumetofen（Miravis）和嘧菌酯（Elatus），弗吉尼亚理工大学，2020年11月。在20世纪90年代初，百菌清以1.2千克ai/公顷的速度用于控制迟发性叶斑病，尽管当与环丙唑联合使用时，剂量可以降低到0.42千克ai/公顷。

. Later, in 2008, it was shown that prothioconazole at 0.18–0.20 kg ai/ha was more effective than tebuconazole 0.23 kg ai/ha in similar regimes of application, and these were more effective than the standard of chlorothalonil 1.2 kg ai/ha

后来，在2008年，研究表明，在类似的应用方案中，0.18-0.20 kg ai/ha的丙硫唑比0.23 kg ai/ha的戊唑醇更有效，并且比百菌清1.2 kg ai/ha的标准更有效

. All these fungicides are registered for their use on peanuts, and are currently applied in various combinations and concentrations.

。所有这些杀菌剂都登记用于花生，目前以各种组合和浓度使用。

A single spore of CP grown in vitro can take six months to form a 5-mm diameter colony which over time displays color and texture variation

体外生长的CP的单个孢子可能需要六个月才能形成直径为5毫米的菌落，随着时间的推移，菌落会显示出颜色和质地的变化

; spores being multicellular, further confound the work with CP. Some tools recently developed will help advance CP research. The first genome and transcriptome of CP isolate NRRL 64,463 have been sequenced and annotated

；孢子是多细胞的，进一步混淆了CP的工作。最近开发的一些工具将有助于推进CP研究。对CP分离株NRRL 64463的第一个基因组和转录组进行了测序和注释

, and image analysis was proven effective to quantify CP growth (

，并且图像分析被证明可以有效地量化CP的增长(

https://imagej.net/ij/

, National Institutes of Health, Bethesda, MD)

，国立卫生研究院，马里兰州贝塞斯达）

, .

Plants have sophisticated sensory systems that activate immune responses when detecting either molecules that are not part of the plant (non self), such as pathogen- or microbe-associated molecular patterns (PAMPs, or MAMPs), or molecules that belong to the plant but are altered, damage-associated molecular patterns (DAMPs).

植物具有复杂的感觉系统，当检测到不属于植物（非自身）的分子时，如病原体或微生物相关分子模式（PAMP或MAMP），或属于植物但被改变的分子，激活免疫反应，损伤相关分子模式（DAMP）。

. Examples of effectors recognized by plants are β-glucans, chitin, ergosterol

植物识别的效应物有β-葡聚糖、几丁质、麦角固醇

, and melanin

和黑色素

; whereas the DAMP response is triggered by products of cell-wall degradation

；而DAMP响应是由细胞壁降解的产物触发的

, and DNA damage caused by genotoxic agents

，以及基因毒性剂引起的DNA损伤

. Plants can distinguish self from non-self extracellular or extranuclear DNA with high taxonomic specificity, and trigger levels of DAMP response based on DNA length accordingly

植物可以区分自身和非自身细胞外或核外DNA，具有很高的分类学特异性，并根据DNA长度相应地触发DAMP反应水平

Overcoming plant resistance for phytopathogenic fungi is a matter of time and reproductive biology of the fungus

克服植物对植物病原真菌的抗性是一个时间问题，也是真菌的生殖生物学问题

. Sexual reproduction, mutation rate, and high genetic diversity all increase the potential of the pathogen to overcome plant resistance

有性生殖、突变率和高遗传多样性都增加了病原体克服植物抗性的潜力

. In Ascomycetes, sexual reproduction is controlled by the mating-type locus (MAT1) which has two idiomorphs, one encoding an alpha-box motif protein (MAT1-1), and the other a High Mobility Group (HMG) (MAT1-2)

在子囊菌中，有性繁殖受交配型基因座（MAT1）控制，该基因座具有两个自形，一个编码α盒基序蛋白（MAT1-1），另一个编码高迁移率族（HMG）（MAT1-2）

. We had reported partial information of the CP MAT1 locus

。我们报告了CP MAT1基因座的部分信息

, however, detailed annotation of both idiomorphs, crucial information to perform population genetic studies, is not yet available.

然而，尚未获得两种习语的详细注释，这是进行种群遗传学研究的关键信息。

Here we analyzed the morphology, genetics, and chemical composition of four different phenotypes of CP isolates to begin understanding the plant offense capability of this pathogen, and the challenge of developing peanut germplasm with LLS resistance.

在这里，我们分析了四种不同表型的CP分离株的形态，遗传学和化学成分，以开始了解这种病原体的植物攻击能力，以及开发具有LLS抗性的花生种质的挑战。

Results

结果

Morphological characterization and growth rate

形态特征和生长速率

The three colored-CP isolates are available at USDA-ARS-NRRL collection, Peoria, IL, accessions NRRL 64,627 for red morphotype (RED), Fig.

三种颜色的CP分离株可在USDA-ARS-NRRL collection，Peoria，IL，种质NRRL 64627中获得红色形态型（红色），图。

a, NRRL 64628 tan morphotype (TAN), Fig.

a、 NRRL 64628 tan形态型（tan），图。

b, and NRRL 64,629 brown morphotype (BROWN), Fig.

b、和NRRL 64629棕色形态型（棕色），图。

c. Conidia production was widespread after 9-day incubation for BROWN and RED, while sporulation was less abundant and took 2–3 weeks in TAN and NRRL 64,463. Orange, extracellular pigments were observed in BROWN and RED, with pigments most frequently occurring as crystals attached to the hyphae in BROWN, and as liquid droplets or crystals in the RED isolate.

c、分生孢子在棕色和红色培养9天后广泛产生，而孢子形成较少，在棕褐色和NRRL 64463中需要2-3周。在棕色和红色中观察到橙色的细胞外色素，色素最常以棕色附着在菌丝上的晶体形式出现，在红色分离物中以液滴或晶体形式出现。

Pigments were not observed in association with hyphae in TAN or NRRL 64,463. RED, TAN and NRRL 64,463 developed some verrucose hyphae. Standard hyphal widths were 5 μm, though verrucose hyphae were ≤ 3.5 μm, mean 2.4 μm, and the bump densities were high, with mean bump diameters at 1 μm, Fig. .

在TAN或NRRL 64463中未观察到与菌丝相关的色素。红色，棕褐色和NRRL 64463形成了一些疣状菌丝。标准菌丝宽度为5μm，尽管疣状菌丝≤3.5μm，平均2.4μm，并且凹凸密度很高，平均凹凸直径为1μm，图。

d-e-f-g-h-i-j. The verrucose structures observed in BROWN had close association with conidia and shape similar to conidiophores rather than hyphae. RED had a moist, rubbery appearance and flexible consistency, Fig.

d-e-f-g-h-i-j.在棕色中观察到的疣状结构与分生孢子密切相关，形状类似于分生孢子而不是菌丝。红色具有潮湿的橡胶状外观和柔韧的稠度，图。

a, compared to the more rigid and dry structure of BROWN and TAN, Fig. 1bc.

a、与棕色和棕褐色更坚硬和干燥的结构相比，图1bc。

Fig. 1

图1

Morphology of three colored CP isolates.

三种颜色CP分离株的形态。

一

b类

c级

: stereoscope view 2 x magnification of NPR22 (RED), NPT22 (TAN) and NPO22 (BROWN), respectively.

：NPR22（红色），NPT22（棕褐色）和NPO22（棕色）的立体镜视图分别放大2倍。

: scanning electron microscopy (SEM) 1000X showing verrucose hyphae in 10-day PDA culture of NRRL 64,463.

：扫描电子显微镜（SEM）1000X显示NRRL 64463的10天PDA培养物中的疣状菌丝。

f级

克

: 10-day PDA culture NPR22, left to right, light microscope 400X highlighting crystals, 1000X highlighting pigment droplets, scanning electron microscopy (SEM) showing verrucose hyphae and crystals; → indicates crystals.

：10天PDA培养NPR22，从左到右，光学显微镜400X突出显示晶体，1000X突出显示色素滴，扫描电子显微镜（SEM）显示疣状菌丝和晶体；→表示晶体。

小时

我

: left to right, SEM showing verrucose structures on 9-day PDA cultures of NPO22, NPR22, and NPT22. Images

：从左到右，SEM显示NPO22，NPR22和NPT22的9天PDA培养物上的疣状结构。图像

一

-（笑声）

c级

were generated by Dr. R. Arias, figures

由R.Arias博士生成

–

were generated by Dr. E. Cantonwine.

由E.Cantonwine博士生成。

Full size image

全尺寸图像

Genome sequencing and analysis of variants

基因组测序和变异分析

The raw reads of the genomic libraries of RED, TAN and BROWN, were uploaded to NCBI-GenBank, SRA accessions SRR24707293, SRR24718870 and SRR24718913, respectively, Supplementary Table 1. Mapping genomic sequencing reads to conserved genes: ribosomal RNA operon, RNA-polymerase II largest subunit (RPB1) and RNA-polymerase II second largest subunit (RPB2), showed that color-CP isolates had 99.95% identity to the reference genome NRRL 64,463.

红色，棕色和棕色基因组文库的原始读数分别上传到NCBI GenBank，SRA种质SRR24707293，SRR24718870和SRR24718913，补充表1。将基因组测序读数映射到保守基因：核糖体RNA操纵子，RNA聚合酶II最大亚基（RPB1）和RNA聚合酶II第二大亚基（RPB2），表明彩色CP分离株与参考基因组NRRL 64463具有99.95%的同一性。

Nucleotide changes in rRNA operon, RPB1 and RPB2 were 0.23, 0.36 and 0.45 nt/1000 nt. One SNP variant in the rRNA operon was observed in all three color-CP isolates and not present in NRRL 64,463. Two SNP variants were detected, one on RPB1 and one on RPB2, Fig. .

rRNA操纵子，RPB1和RPB2的核苷酸变化分别为0.23、0.36和0.45 nt/1000 nt。在所有三种颜色的CP分离株中均观察到rRNA操纵子中的一个SNP变体，而在NRRL 64463中不存在。检测到两个SNP变体，一个在RPB1上，一个在RPB2上，图。

a, not present in the published reads of NRRL 64,463, Fig.

a、 NRRL 64463的公开阅读中不存在，图。

答：。

Fig. 2

图2

Genomic variants and RNA sequencing summary of three color

三色基因组变异和RNA测序概述

Cercosporidium personatum

人尾孢霉

(CP) isolates.

（CP）分离株。

一

: Annotation of CP ribosomal RNA operon (rRNA), and nucleotide variants in rRNA, largest and second largest subunits of RNA Polymerase II (RPB1 and RPB2).

：注释CP核糖体RNA操纵子（rRNA）和rRNA中的核苷酸变体，RNA聚合酶II的最大和第二大亚基（RPB1和RPB2）。

b类

: Genomic variants in three color CP isolates with ≥ 35% frequency not detected in the reference CP genome NRRL 64,463, top number: total single- and multi-nucleotide polymorphisms (SNP, MNP), [ ]: number of insertions, ( ): number of deletions.

：在参考CP基因组NRRL 64463中未检测到频率≥35%的三色CP分离株的基因组变异，顶部编号：总单核苷酸和多核苷酸多态性（SNP，MNP），[]：插入数，（）：缺失数。

c级

: Principal Component Analysis (PCA) of eight RNA sequencing libraries.

：八个RNA测序文库的主成分分析（PCA）。

: Number of predicted amino acid changes (AAC) on secondary metabolite biosynthesis gene clusters detected by antiSMASH 5.0 reported in

：antiSMASH 5.0检测到的次级代谢产物生物合成基因簇的预测氨基酸变化（AAC）数量

https://doi.org/10.7910/DVN/RMBQ0E

(Arias et al. 2023), ↓: no AAC predicted.

（Arias等人，2023年），↓: 没有AAC预测。

Full size image

全尺寸图像

Mapping colored-CP genome sequencing reads to the 1061 contigs of NRRL 64,463 genome resulted in an average coverage of 140 X, 61 X, and 68 X, for RED, TAN and BROWN CP isolates, respectively. Genome-wide variants using 1% cutoff in all four isolates showed most variants had frequencies higher than 90% or lower than 10%, Supplementary Fig. 2.

将彩色CP基因组测序读数映射到NRRL 64463基因组的1061个重叠群，导致红色，棕褐色和棕色CP分离株的平均覆盖率分别为140倍，61倍和68倍。在所有四个分离株中使用1%截止值的全基因组变体显示大多数变体的频率高于90%或低于10%，补充图2。

Using 35% cutoff, most variants were single and multi-nucleotide polymorphisms (SNP, MNP), accounting for 92% in TAN, 91% in RED, and 88% in BROWN. Variants of heterozygous loci (2 or more nt changes) were 69% in TAN and RED, and 80% in BROWN. Genomic variants observed with a ≥ 35% cutoff in the reference genome NRRL 64,463 were subtracted from variants of the same percentage cutoff in RED, TAN and BROWN, Supplementary Table 2.

使用35%的截止值，大多数变体是单核苷酸和多核苷酸多态性（SNP，MNP），棕褐色占92%，红色占91%，棕色占88%。杂合基因座的变异（2个或更多nt变化）在棕褐色和红色中为69%，在棕色中为80%。从红色，棕褐色和棕色的相同截止百分比的变体中减去在参考基因组NRRL 64463中观察到的截止值≥35%的基因组变体，补充表2。

The resulting variants were largest in BROWN (32,966), from which 16,455 were shared by all three colored-CP isolates, Fig. .

由此产生的变体在棕色中最大（32966），其中16455个由所有三种颜色的CP分离株共享，图。

b. Despite the abundance of variants, all three colored-CP isolates had 99.9% identity to the CP reference genome, confirming their taxonomic identification. Mapping of reads to CBS 151044 chromosomes indicated parts of the 5.7 Mbp-chromosome 6 were missing in the isolates as follows: 26% (1.5 Mbp), 16% (910 kb), 14% (803 kb) and 7% (386 kb) in NRRL 64,463, TAN, RED and BROWN, respectively..

b、尽管变体丰富，但所有三种颜色的CP分离株与CP参考基因组具有99.9%的同一性，证实了它们的分类学鉴定。将读数映射到CBS 151044染色体表明，分离株中缺失了5.7 Mbp染色体6的部分，如下所示：NRRL 64463，棕褐色，红色和棕色分别为26%（1.5 Mbp），16%（910 kb），14%（803 kb）和7%（386 kb）。。

RNA sequencing (RNAseq) and secondary metabolites

RNA测序（RNAseq）和次级代谢产物

RNAseq

RNASEq

mRNAs from two biological replicates of 12-day old cultures of RED, BROWN, TAN and NRRL 64,463, grown with photoperiod 16/8 h light/darkness were sequenced. A list of sequencing reads and accession numbers at NCBI are in Supplementary Table 3. A principal component analysis (PCA) performed to visualize a summary and quality of the results.

对在光周期16/8小时光照/黑暗条件下生长的12天大的红色，棕色，棕褐色和NRRL 64463培养物的两个生物学重复的mRNA进行测序。NCBI的测序读数和登录号列表见补充表3。进行主成分分析（PCA）以可视化结果的摘要和质量。

showed congruence between replicate samples, and a 92.5% of the variation being explained by the first four dimensions, Fig.

显示重复样本之间的一致性，92.5%的变异由前四个维度解释，图。

c. Overall, 5,201 (48%) of the 10,781 CP genes were differentially expressed on the four CP isolates based on a Bonferroni corrected probability

c、总体而言，根据Bonferroni校正的概率，10781个CP基因中的5201个（48%）在四个CP分离株上差异表达

≤ 1.0E-5, 60 of those genes had ≥ 100-fold change. After mapping reads to secondary metabolite gene clusters, predicted amino acid changes (AAC) was highest in BROWN and lowest in TAN, Fig.

≤1.0E-5，其中60个基因的变化≥100倍。将读数映射到次级代谢产物基因簇后，预测的氨基酸变化（AAC）在棕色中最高，在棕褐色中最低，图。

d. Based on the annotated reference genome NRRL 64,463

d、基于注释的参考基因组NRRL 64463

we found significant differential expression (

我们发现了显著的差异表达(

≤ 10

− 5

−5

) of 41 dbCAN identified genes and 47 PHI-BLAST genes. Heat maps of these results displayed clear patterns between isolates and high uniformity between replicates, Supplementary Figs. 3, 4. Also 192 apoplastic or cytoplasmic genes predicted by Effector-P were differentially expressed (

)在41个dbCAN鉴定的基因和47个PHI-BLAST基因中。这些结果的热图显示了分离株之间的清晰模式和重复之间的高度均匀性，补充图3,4。效应蛋白预测的192个质外或细胞质基因也有差异表达(

< 10

− 5

−5

) with changes between 2 and 8433-fold.

)变化在2到8433倍之间。

Dothistromin/Aflatoxin biosynthesis gene mini-clusters

Dothistromin/黄曲霉毒素生物合成基因微簇

Genes functionally confirmed, co-regulated or putatively involved in dothistromin (DOT) or aflatoxin (AF) biosynthesis in

基因在功能上被证实，共同调节或推测参与了dothistromin（DOT）或黄曲霉毒素（AF）的生物合成

Dothistroma

多特罗马症

spp. or

spp.或

Aspergillus

曲霉

spp

spp公司

. were BLAST to

.被炸到

Cladosporium fulvum

黄枝孢菌

(NCBI:

（NCB：

JARJJH000000000

) to which CP showed higher homology than to

)CP与之显示出比与之更高的同源性

Dothistroma

多特罗马症

spp. The resulting hits were BLAST to the CP reference genome NRRL 64,463

结果命中CP参考基因组NRRL 64463

, and annotated contigs were mapped to the CP chromosomes

，并将带注释的重叠群定位到CP染色体上

. All genes identified, including the transcription activator

.鉴定出的所有基因，包括转录激活因子

aflR

, mapped to chromosome 12, except for averantin hydroxylase (

，定位于12号染色体，除了阿维菌素羟化酶(

AvnA

) of which two copies were found in chromosomes 4 and 10 (Supplementary Table 4). DOT biosynthesis genes were observed in mini clusters within contigs 469, 242, 291, 472, and 784 of NRRL 64,463, their representation at scale is shown in Fig.

)其中在染色体4和10中发现了两个拷贝（补充表4）。在NRRL 64463的重叠群4692429291472和784中的小簇中观察到点生物合成基因，它们的规模表示如图1所示。

. A complete list of the genes identified in the DOT biosynthesis pathway and their expression levels on CP isolates is shown in Supplementary Fig. 5. Differential expression of transcripts annotated as part of the DOT biosynthesis pathway was evaluated and their predicted gene products later confirmed by LC-MS, Fig. .

补充图5显示了在DOT生物合成途径中鉴定的基因及其在CP分离株上的表达水平的完整列表。评估了注释为DOT生物合成途径一部分的转录本的差异表达，并随后通过LC-MS证实了其预测的基因产物，图。

. Overall, the highest level of expression of these genes was observed in BROWN and the lowest in RED, Fig.

总体而言，这些基因的表达水平最高的是棕色，最低的是红色，图。

. Analysis of gene expression by qPCR of five genes in the DOT pathway showed similar trends compared to the RNAseq data, except in TAN, for which results were inconsistent, Fig.

通过qPCR分析DOT途径中五个基因的基因表达显示出与RNAseq数据相似的趋势，除了TAN，其结果不一致，图。

Fig. 3

图3

Genes involved in the dothistromin biosynthesis pathway. Scale annotation of mini-clusters in contigs, and contig positions on chromosome 12 of

参与dothistromin生物合成途径的基因。重叠群中小簇的规模注释，以及12号染色体上的重叠群位置

Cercosporidium personatum

人尾孢霉

. Contig_298 harbors the mating type (MAT) locus. Contig numbers from Arias et al.. 2023

重叠群298具有交配型（MAT）基因座。Arias等人2023年的重叠群数字

, chromosome reference published in Gonzales et al. 2024

，染色体参考发表在Gonzales等人的2024年

, . Mbp: mega basepairs.

。Mbp：兆碱基对。

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Fig. 4

图4

Dothistromin biosynthesis pathway gene expression in four

Dothistromin生物合成途径基因在四种细胞中的表达

Cercosporidium personatum

人尾孢霉

(CP) isolates.

（CP）分离株。

Genes

基因

: orthologous genes from

：来自

Fulvia fulva

番茄叶霉病菌

Dothistroma

多特罗马症

spp and

spp和

Aspergillus

曲霉

spp. found in CP using the gene models of NRRL 64,463 (Arias et al. 2023)

使用NRRL 64463的基因模型在CP中发现的物种（Arias等人，2023）

Gene Expression

基因表达

: Log

：日志

of the fold change, probability level with Bonferroni correction (

折叠变化的概率水平与邦弗朗尼校正(

< 1.0E-5).

<1.0E-5）。

Products

产品

: Predicted products for each group of genes and identified in chemical analysis: NOR: norsolorinic acid, AVN: averantin, AVR: averufin, VERB: versicolorin B, VERA: versicolorin A.

：每组基因的预测产物，并在化学分析中鉴定：NOR：去甲索酸，AVN：averantin，AVR：averufin，动词：versicolorin B，VERA：versicolorin A。

qPCR

: results of quantitative Real-Time PCR for some genes in the pathway.

：该途径中某些基因的定量实时PCR结果。

: indicates that dothistromin-related compounds are the expected products, though they are presumed present, their identification will require additional work.

：表明与dothistromin相关的化合物是预期的产物，尽管推测它们存在，但其鉴定将需要额外的工作。

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Quantitation of anthraquinones (AQ)

蒽醌（AQ）的定量

Here we report for the first time in

在这里，我们首次报道

Cercosporidium personatum

人尾孢霉

the following AQ being produced: norsolorinic acid (NOR), averantin (AVN), averufin (AVR), averufanin (AVF), nidurufin (NID), versicolorin B (VERB), and versicolorin A (VERA), Fig.

产生以下AQ:norsolorinic acid（NOR），averantin（AVN），averufin（AVR），averufanin（AVF），nidurufin（NID），versicolorin B（动词）和versicolorin A（VERA），图。

. These compounds were identified by comparison with authentic standards, and all were detected in the four CP isolates studied. The region marked as

通过与真实标准品比较鉴定了这些化合物，并且在所研究的四种CP分离株中均检测到。标记为的区域

in the chromatogram, corresponds to dothistromin-related compounds, Fig.

在色谱图中，对应于dothistromin相关化合物，图。

. Metabolites that were searched and not found in the samples, based on comparison with authentic standards and within the detection limits of the analysis were: versiconol, versiconol acetate, aversin, ortho-methylsterigmatocystin (OMST), sterigmatocystin (ST), aflatrem, dihydroxyaflavinine, emodin, dihydro ST, dihydro OMST, asparason A, aflatoxins B.

根据与真实标准的比较，在分析的检测限内，在样品中未发现的代谢物是：versiconol，versiconol acetate，aversin，邻甲基杂色曲霉素（OMST），杂色曲霉素（ST），黄曲霉素，二羟基黄曲霉素，大黄素，二氢ST，二氢OMST，天冬氨酸A，黄曲霉毒素B。

, B

，B

, G

，克

, G

，克

, M

，米

and B

和B

2a级

. The metabolites present in the fungal biomass of the four CP isolates concurred with the levels of expression of DOT-biosynthesis genes. Significantly higher levels of expression and actual concentrations of NOR (468 µg/g Dry Weight, DW), AVN (1704), AVR (8685), VERB (1779) and VERA (401) were present in BROWN, and the lowest concentrations were observed in RED, with NOR (60 µg/g), AVN (249), AVR (6098), VERB (454), VERA (116), Fig. .

。四种CP分离株的真菌生物量中存在的代谢物与DOT生物合成基因的表达水平一致。棕色中存在显着更高水平的NOR（468µg/g干重，DW），AVN（1704），AVR（8685），VERB（1779）和VERA（401）的表达和实际浓度，最低浓度为红色，NOR（60µg/g），AVN（249），AVR（6098），VERB（454），VERA（116），图。

. Overall, 7-8-fold more NOR and AVN, and 3-4-fold more VERB and VERA were observed in BROWN compared to RED morphotype, Fig.

总体而言，与红色形态相比，棕色中观察到的NOR和AVN多7-8倍，动词和VERA多3-4倍。

. Two other AQ, Averufanin (AVF) and Nidurufin (NID), were also present in the CP isolates, though these are not precursors in the DOT/AF biosynthesis pathway

CP分离株中还存在另外两种AQ，阿维鲁法宁（AVF）和尼杜鲁芬（NID），尽管它们不是DOT/AF生物合成途径中的前体

. The highest concentration of AVF was present in TAN (51 µg/g) and the highest NID was present in NRRL 64,463 with almost 1 mg/g (929 µg/g DW).

。最高浓度的AVF存在于TAN中（51µg/g），最高NID存在于NRRL 64463中，几乎为1 mg/g（929µg/g DW）。

Fig. 5

图5

Typical chromatogram observed for

观察到的典型色谱图

Cercosporidium personatum

人尾孢霉

(CP) grown for 12 days with photoperiod 16/8 h light/darkness. Image generated by Dr. V. Sobolev.

（CP）在光周期16/8小时光照/黑暗下生长12天。图像由V.Sobolev博士生成。

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Fig. 6

图6

Anthraquinones and ergosterol that were identified and quantitated in four isolates of

在四个分离株中鉴定和定量的蒽醌和麦角固醇

Cercosporidium personatum

人尾孢霉

(CP) using five replicates of each culture grown for 12 days.

（CP）使用生长12天的每种培养物的五个重复。

Light

灯光

: photoperiod 16/8 h light/dark,

：光周期16/8小时亮/暗，

Dark

深色

: incubation in the dark (shaded area). Precursors in the dothistromin/aflatoxin pathway are connected by arrows, NOR: norsolorinic acid, AVN: averantin, AVR: averufin, VERB: versicolorin B, VERA: versicolorin A. Compounds related to the pathway but not direct precursors of aflatoxin are AVF: averufanin and NID: nidurufin.

：在黑暗中孵育（阴影区域）。dothistromin/黄曲霉毒素途径中的前体通过箭头连接，NOR：去甲索酸，AVN：averantin，AVR：averufin，动词：versicolorin B，VERA：versicolorin A.与该途径相关但不是黄曲霉毒素直接前体的化合物是AVF：averufanin和NID：nidurufin。

Also graphed is the concentration of ergosterol (ERG) that was detected in the analyses. Different letters on the bars of the histograms, within light conditions separately from dark conditions, indicate statistically significant differences, Tukey’s test (.

还绘制了分析中检测到的麦角固醇（ERG）浓度。直方图条上的不同字母，在光照条件下与黑暗条件下分开，表明统计学上的显着差异，Tukey检验（。

< 0.05).

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Ergosterol

麦角固醇

LC-MS analysis detected ergosterol. In cultures grown 12 days with 16/8 h photoperiod and 26 °C, significantly higher ergosterol content (

LC-MS分析检测到麦角固醇。在光周期为16/8小时，温度为26°C的培养物中，麦角固醇含量明显较高(

< 0.01) was observed in RED and TAN (743, 792 µg/g DW, respectively), Fig.

在红色和棕色（分别为743792µg/g DW）中观察到<0.01）。

. Concurrently, levels of expression of six ergosterol biosynthesis genes showed correlation with the abundance of ergosterol under photoperiod, Fig.

同时，六种麦角固醇生物合成基因的表达水平与光周期下麦角固醇的丰度相关，图。

. Isolates BROWN and CP had the lowest ergosterol content when grown in photoperiod (100 and 200 µg/g), though showed an ~ 18-fold increase (2000 and 4000 µg/g, respectively) when grown in darkness, Fig.

分离株BROWN和CP在光周期（100和200µg/g）中生长时麦角固醇含量最低，但在黑暗中生长时，麦角固醇含量增加了约18倍（分别为2000和4000µg/g）。

. RED grown in darkness reached 4341 µg/g.

在黑暗中生长的红色达到4341微克/克。

Fig. 7

图7

Differential gene expression of ergosterol biosynthesis genes, chitin-synthases, chitinases, and melanin biosynthesis genes in four

麦角固醇生物合成基因、几丁质合成酶、几丁质酶和黑色素生物合成基因在四种基因中的差异表达

Cercosporidium personatum

人尾孢霉

(CP) isolates. Orthologous genes involved in the biosynthesis of these compounds, and found to have significantly different levels of expression using Bonferroni correction

（CP）分离株。与这些化合物的生物合成有关的直系同源基因，通过邦弗朗尼校正发现其表达水平存在显著差异

< 1.0E-5 are represented. Quantification of β-[1,3] and β-[1,4] glucans and melanin are shown in histograms where different letters on the bars indicate statistically significant differences, Tukey’s test (

表示<1.0E-5。图中显示了β-[1,3]和β-[1,4]葡聚糖和黑色素的定量，其中条形图上的不同字母表示统计学上的显着差异，Tukey检验(

< 0.05). qRT-PCR: quantitative real-time PCR for two genes involved in melanin biosynthesis, CP: reference isolate NRRL 64,463.

<0.05）。qRT-PCR：对参与黑色素生物合成的两个基因进行定量实时PCR，CP：参考分离株NRRL 64463。

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Chitin, β-[1,3] and β-[1,4] glucans

甲壳素、β-[1,3]和β-[1,4]葡聚糖

Most fungal cell walls have a central core of β-[1,3], [1,8] glucan anchored to chitin via a β-[1,4] linkage

大多数真菌细胞壁的中心核心是通过β-[1,4]键锚定在几丁质上的β-[1,3]、[1,8]葡聚糖

. Calcofluor white binds to β-[1,3] and β-[1,4] polysaccharides of chitin and cellulose and was used here to quantify these compounds in the isolates. The content of β-[1,3] and β-[1,4] glucans in the isolates was 4.320, 0.856, 0.264 and 0.116 mg/100 mg DW, for RED, TAN, BROWN and NRRL 64463, respectively, and the differences were statistically significant (.

Calcofluor white与几丁质和纤维素的β-[1,3]和β-[1,4]多糖结合，并用于定量分离物中的这些化合物。对于红色，棕褐色，棕色和NRRL 64463，分离株中β-[1,3]和β-[1,4]葡聚糖的含量分别为4.320、0.856、0.264和0.116 mg/100 mg DW，差异有统计学意义（。

≤ 1.0E-2), Fig.

≤1.0E-2），见图。

. However, RED had the lowest levels of expression of four chitin synthases explored, and TAN had the highest, whereas two chitinases with differential expression (

然而，红色的四种几丁质合酶的表达水平最低，而TAN的表达水平最高，而两种几丁质酶的表达水平不同(

< 1.0E-5), had opposite levels of expression in RED, Fig.

<1.0E-5），红色表达水平相反，图。

. Among differentially expressed carbohydrate active enzymes (dbCAN), 11 genes were highly expressed in RED and had low expression in other isolates, Supplementary Fig. 3.

在差异表达的碳水化合物活性酶（dbCAN）中，11个基因以红色高度表达，而在其他分离株中表达较低，补充图3。

Melanin

黑色素

Melanin content in the isolates was quantified using published methods

使用已发表的方法定量分离株中的黑色素含量

, after extracting AQ. Some genes involved in melanin-biosynthesis are polyketide synthase 1 (

，提取AQ后。一些参与黑色素生物合成的基因是聚酮合酶1(

pks1

)

, scytalone dehydratase (

，单体脱水酶(

Scd1

)

, hydroxynaphthalene reductase-like (

，羟基萘还原酶样(

Arp2

Arp2类

), tetra-hydroxynaphthalene reductase (

)，四羟基萘还原酶(

Thnr

Thnr公司

)

. These were differentially expressed across the four CP isolates, with highest expression levels observed in BROWN in RNAseq (

。这些在四个CP分离株中差异表达，在RNAseq的棕色中观察到最高的表达水平(

< 1.0E-5), confirmed by qPCR (

<1.0E-5），经qPCR证实(

≤ 1.0E-3) and correlated with the highest melanin content present in this isolate, 14.8 mg/g (

≤1.0E-3），并与该分离物中最高的黑色素含量14.8 mg/g相关(

≤ 1.0E-3). Up-regulation of

≤1.0E-3）。上调

pks1

Thnr

Thnr公司

Arp2

Arp2类

and

和

Scd1

in BROWN was 372-, 160-, 30- and 21-fold, respectively, whereas RED had the lowest melanin content (6.3 mg/g), and lowest expression of these genes in RNAseq and qPCR, Fig.

棕色分别为372倍，160倍，30倍和21倍，而红色的黑色素含量最低（6.3 mg/g），这些基因在RNAseq和qPCR中的表达最低。

Pathogenicity

致病性

All four CP isolates caused typical leafspot lesions in two peanut cultivars, Georgia-06G and AU-NPL17. Based on 24 inoculation sites, 4–50% of the sites presented symptoms in Georgia-06G, and 8–46% in AU-NPL17. The largest number of lesions were observed on different isolates in repeated experiments, thus, there was not sufficient evidence to consider any more virulent than others based on the detached-leaf assay..

所有四种CP分离株在两个花生品种Georgia-06G和AU-NPL17中引起典型的叶斑病变。根据24个接种地点，乔治亚州06G有4-50%的地点出现症状，AU-NPL17有8-46%的地点出现症状。在重复实验中，在不同分离株上观察到的病变数量最多，因此，没有足够的证据表明基于离体叶片测定法，其毒性比其他分离株更强。。

Mating type

配合类型

We described for the first time the CP MAT1 locus, including MAT1-1 and MAT1-2 genes orthologous to those in

我们首次描述了CP MAT1基因座，包括MAT1-1和MAT1-2基因与

Cladosporium fulvum

黄枝孢菌

Race 5

比赛5

. CP contig_298 was located on chromosome 12, flanked by DOT biosynthesis gene mini clusters, Fig.

CP重叠群298位于12号染色体上，两侧是点生物合成基因小簇，图。

. A dot plot of NRRL 64463 contig_298

NRRL 64463重叠群298的点图

与

. the orthologous region in CBS 151044 showed a gene inversion around position 35 kb, on the MAT + sexual cell fertilization-promoting factor (augustus_masked-contig_298-processed-gene-0.26), labelled as MAT1-2 in the colored CP isolates and NRRL 64463, Supplementary Fig. 6. We found that all three colored CP isolates studied here and the reference isolate NRRL 64463 harbor the MAT1-2 gene, whereas isolates IPAVE 0302.

CBS 151044中的直系同源区域在MAT + 性细胞受精促进因子（augustus u masked-contig u 298-processed-gene-0.26）上显示出35 kb左右的基因倒置，标记为MAT1-2在有色CP分离株和NRRL 64463中，补充图6。我们发现这里研究的所有三种有色CP分离株和参考分离株NRRL 64463都含有MAT1-2基因，而分离株IPAVE 0302。

and CBS 151044

和CBS 151044

harbor the MAT1-1 gene, Supplementary Fig. 7. Reverse primers designed for MAT1-1 and MAT1-2 of

携带MAT1-1基因，补充图7。为MAT1-1和MAT1-2设计的反向引物

Cladosporium fulvum

黄枝孢菌

, (MAT1-1 P4R 5’-TGTTCGGTGTCGTGATG-3’, MAT1-2 P4R 5’-TCCACGTCGAAGTAGAG-3’)

，（MAT1-1 P4R 5'-TGTTCGGTGTCGTGATG-3'，MAT1-2 P4R 5'-TCCACGTCGAAGTAGAG-3'）

were both found in CP MAT1 locus, but the forward primers did not match. Hence, we designed forward primers: MAT1-1_CPFw 5’-TCCTGTGGCGTGCCGATCCC-3’, and MAT1-2_CPFw 5’-TACCCACCGACCATGCTATC-3’, that in combination with the reverse primers reported

均在CP MAT1基因座中发现，但正向引物不匹配。因此，我们设计了正向引物：MAT1-1\u CPFw 5'-TCCTGTGGCGATCCC-3'和MAT1-2\u CPFw 5'-TACCACCGACCATGCTATC-3'，与报道的反向引物结合使用

will amplify a 759 bp and a 997 bp fragments of idiomorphs MAT1-1 and MAT1-2, respectively.

将分别扩增自形MAT1-1和MAT1-2的759 bp和997 bp片段。

Response to xenobiotics

对异生素的反应

Overall, the three color-CP isolates had higher levels of resistance to chlorothalonil (CHLOR), tebuconazole (TEB), and caffeine (CAF) compared to the reference isolate NRRL 64,463 (

总体而言，与参考分离株NRRL 64463相比，这三种颜色的CP分离株对百菌清（CHLOR），戊唑醇（TEB）和咖啡因（CAF）的抗性水平更高(

< 0.05), whereas TAN was slightly more susceptible to prothioconazole (PROT) (

<0.05），而TAN对丙硫环唑（PROT）的敏感性稍高(

< 0.05), Supplementary Fig. 8. The recommended inoculum size, 5-day incubation and the use of PVDF membranes were suitable to test CP fungicide resistance, Fig.

<0.05），补充图8。推荐的接种量，5天孵育和PVDF膜的使用适合于测试CP杀菌剂的抗性，图。

. Average dose of xenobiotics that inhibit growth by 50% (EC

。抑制生长50%的异生素的平均剂量（EC

)

were calculated for two experiments using AAT Bioquest for four parameters

使用AAT Bioquest计算了两个实验的四个参数

, and were: CHLOR 1.06 mg/L, PROT 1.56 mg/L, TEB 18.22 mg/L, and CAF 7930 mg/L (40.84 mM). Both PROT and TEB are triazole fungicides, and since changes in the sterol 14-α demethylase (cyp51) gene are usually associated to azole-fungicide resistance

，分别为：氯1.06 mg/L，PROT 1.56 mg/L，TEB 18.22 mg/L和CAF 7930 mg/L（40.84 mM）。PROT和TEB都是三唑类杀菌剂，由于固醇14-脱甲基酶（cyp51）基因的变化通常与唑类杀菌剂的耐药性有关

, we performed BLAST search for this gene in the reference CP transcriptome. Gene sm_ctg_825 − 0.1 corresponded to sterol 14-α demethylase and was up-regulated (

，我们在参考CP转录组中对该基因进行了BLAST搜索。sm ctg U 825- 0.1基因对应于固醇14-α脱甲基酶，并被上调(

< 10E

<10E

− 10

−10

) by 23-, 8- and 3-fold in RED, TAN and BROWN, respectively. BLAST search identified 11 glutathione S-transferase (GST) genes (E < 10

)红色，棕褐色和棕色分别增加了23倍，8倍和3倍。BLAST搜索确定了11个谷胱甘肽S-转移酶（GST）基因（e10

− 4

) in the CP transcriptome, nine were differentially expressed (

)在CP转录组中，有9个差异表达(

< 10

− 10

−10

), and eight of those nine had highest expression in TAN, up to 20-fold more than other isolates, whereas the lowest expression occurred in NRRL 64,463. The level of resistance to CHLOR depicted in the histogram for 3.3 and 10 mg/L, Supplementary Fig. 8, correlated with GST expression levels on CP isolates..

)，其中9个在TAN中表达最高，比其他分离株高20倍，而在NRRL 64463中表达最低。直方图中描述的3.3和10 mg/L的氯仿抗性水平（补充图8）与CP分离株的GST表达水平相关。。

Fig. 8

图8

Response of four

四人的回应

Cercosporidium personatum

人尾孢霉

(CP) isolates to xenobiotics. First and last column correspond to the controls (Ctrl) on potato-dextrose agar medium at 0- and 5-days incubation. PROT, TEB and CHLOR are images obtained after 5 days at 10 mg a.i./L of prothioconazole, tebuconazole and chlorothalonil, respectively. CAF are images after 5 days at 10 mM caffeine.

（CP）分离到异生素。第一列和最后一列对应于孵育0天和5天的马铃薯葡萄糖琼脂培养基上的对照（Ctrl）。PROT，TEB和CHLOR分别是在10 mg a.i./L的丙硫唑，戊唑醇和百菌清中5天后获得的图像。CAF是在10 mM咖啡因下5天后的图像。

Images were generated by Dr. R. Arias..

图像由R.Arias博士生成。。

Full size image

全尺寸图像

Discussion

讨论

Single spore isolates of

单孢子分离株

Cercosporidium personatum

人尾孢霉

(CP) often show irregular morphology in culture

（CP）在培养中经常表现出不规则的形态

. Here we explored some probable causes of variations in four CP isolates to help understand the pathogen/host interaction. We first confirmed all four isolates were a single taxon, as highly conserved genes, RPB1, RPB2, rRNA operon, each showed ≤ 1 nt difference in colored CP when compared to NRRL 64,463, Fig. .

在这里，我们探讨了四种CP分离株变异的一些可能原因，以帮助了解病原体/宿主的相互作用。我们首先证实所有四个分离株都是一个单一的分类群，因为与NRRL 64463相比，高度保守的基因RPB1，RPB2，rRNA操纵子在有色CP中均显示出≤ 1 nt的差异，图。

a, and genome-wide SNP rates (1.4–1.7 SNP/kb) granted a 99.9% identity, Fig.

a、全基因组SNP率（1.4-1.7 SNP/kb）赋予99.9%的同一性，图。

b. In comparison, isolates of the plant pathogen

b、相比之下，植物病原体的分离株

Puccinia triticina

小麦锈病

, had rates of 2.4 SNP/kb

，比率为2.4 SNP/kb

, whereas rates of 3.8–5.0 SNP/kb were observed among L-morphotype isolates of

，而在L型分离株中观察到3.8–5.0 SNP/kb的比率

Aspergillus flavus

黄曲霉

. In addition, the higher number of genomic variants at > 90% and < 10% frequencies (Supplementary Fig. 2) were consistent with a homokaryotic distribution

此外，频率>90%和<10%的基因组变异数量较高（补充图2）与同核分布一致

Anthraquinones (AQ) biosynthesis genes and metabolites. CP and

蒽醌（AQ）生物合成基因和代谢物。CP和

C. arachidicola

C.花生

(CA) are phylogenetically closely related

（CA）在系统发育上密切相关

. Since CA can produce dothistromin (DOT)

由于CA可以产生dothistromin（DOT）

, a toxin originally described in

，一种最初在

Dothistroma septosporum

败血性斑点组织瘤

, we speculated DOT could be present in CP. In

，我们推测DOT可能存在于CP中

D. septosporum

D.败血症

, DOT biosynthesis genes are organized in mini-clusters

，点生物合成基因被组织成小簇

, we identified orthologous genes in CP also organized in mini-clusters, Fig.

，我们在CP中鉴定了直系同源基因，这些基因也组织在小簇中，图。

. These co-located to chromosome 12 and had several gene duplications in other chromosomes. Such duplications were also reported in

。这些基因共位于12号染色体上，在其他染色体上有几个基因重复。这种重复也有报道

D. septosporum

D.败血症

. Despite grown in the same conditions, each CP isolate showed contrasting patterns of gene expression throughout their genomes, Figs.

尽管在相同的条件下生长，但每个CP分离株在其整个基因组中显示出不同的基因表达模式。

and

和

, Supplementary Figs. 3, 4, and different concentrations of secondary metabolites, Figs.

，补充图3,4和不同浓度的次级代谢产物，图。

and

和

. AQ such as NOR, AVN and AVR

.AQ，例如NOR、AVN和AVR

, are precursors of the mycotoxins DOT/AF, which share the same pathway between Acetate and VERA

，是真菌毒素DOT/AF的前体，它们在乙酸盐和维拉之间具有相同的途径

, Figs.

，图。

and

和

. With each step of the pathway, the compounds become more toxic. AVF and NID are not directly involved in the biosynthesis of DOT/AF; AVF results from artificial dehydration of hydroxyaverantin (HAVN)

.随着该途径的每一步，化合物的毒性都会增加。AVF和NID不直接参与DOT/AF的生物合成；羟阿维菌素（HAVN）人工脱水导致AVF

, whereas a small fraction of HAVN, in the presence of NADPH or NADP gets converted to NID

，而在存在NADPH或NADP的情况下，一小部分HAVN会转化为NID

. NID, AVN and AVR have shown antimicrobial activity against Gram positive bacterial pathogens with minimum inhibitory doses (MIC) of 2.3, 4.4 and 10.0 µg/mL, respectively

NID，AVN和AVR对革兰氏阳性细菌病原体显示出抗菌活性，最小抑制剂量（MIC）分别为2.3、4.4和10.0µg/mL

. Based on fresh weight, our CP isolates, under photoperiod, accumulated 8-, 9-, and 39-fold above MIC values (19, 42 and 394 µg/mL) for AVN, NID and AVR, respectively, Fig.

根据鲜重，我们的CP分离株在光周期下分别比AVN，NID和AVR的MIC值（19,42和394µg/mL）高8倍，9倍和39倍。

. It would be interesting to examine if these compounds affect the plant microbiome and susceptibility to LLS disease.

研究这些化合物是否影响植物微生物组和对LLS疾病的易感性将是有趣的。

Genotoxicity, or DNA damage in plants, is usually associated to abiotic stresses

植物的遗传毒性或DNA损伤通常与非生物胁迫有关

. However, pathogen effectors can damage DNA and trigger DNA damage response (DDR), damage repair, and in severe cases lead to apoptosis

然而，病原体效应物可以破坏DNA并引发DNA损伤反应（DDR），损伤修复，严重时会导致细胞凋亡

. Whereas NOR and NID have no genotoxicity, from AVR, VERB to VERA the genotoxicity increases

尽管NOR和NID没有遗传毒性，但从AVR，动词到维拉，遗传毒性都会增加

. Based on fresh weight, under photoperiod CP isolates produced 27 to 93 µM of VERA. As a comparison, colon cancer cells exposed to 1 µM of VERA experienced differential expression of 18,000 genes, whereas only 869 genes were differentially expressed with 1 µM aflatoxin B

根据鲜重，在光周期下CP分离株产生27至93µM的VERA。相比之下，暴露于1µM VERA的结肠癌细胞经历了18000个基因的差异表达，而只有869个基因与1µM黄曲霉毒素B差异表达

(AFB1); and VERA triggered apoptosis, showing 10-fold more genotoxicity and four-fold more oxidative stress than AFB

（AFB1）；和VERA触发的细胞凋亡，显示出比AFB高10倍的遗传毒性和四倍的氧化应激

. Isolate BROWN had the highest accumulation of NOR, AVN, AVR, VERB and VERA, under photoperiod and darkness, Fig.

在光周期和黑暗条件下，分离出的棕色具有最高的NOR，AVN，AVR，VERB和VERA积累。

. VERB and VERA possess a bis furan moiety which binds to DNA conferring mutagenic activity

动词和维拉具有双呋喃部分，该部分与赋予诱变活性的DNA结合

, the furan ring causes DNA damage and chromosomal aberrations

，呋喃环会导致DNA损伤和染色体畸变

. Exposure of ten types of cancer cells to AVF, NID, and AVR resulted in the following IC

。将十种类型的癌细胞暴露于AVF，NID和AVR会导致以下IC

, AVF: 11 µM, NID: 40 µM, AVR: 26 µM, in addition AVF caused apoptosis and cell cycle arrest at G1 phase

，AVF:11µM，NID:40µM，AVR:26µM，此外，AVF在G1期引起细胞凋亡和细胞周期停滞

. Based on fresh weight, our CP isolates had AVF concentrations close to IC

根据鲜重，我们的CP分离株的AVF浓度接近IC

, both in photoperiod and darkness (1–14 µM), and NID concentrations in photoperiod (108–189 µM) or darkness (0.2–216 µM), 3-5-fold above its IC

，在光周期和黑暗（1-14m）中，NID浓度在光周期（108-189m）或黑暗（0.2-216 M）中，比其IC高3-5倍

. Overall, if considering growth in photoperiod, BROWN produced higher concentration of compounds known to be genotoxic (AVR, VERB and VERA), whereas the other three isolates produced more of the compounds known to induce apoptosis (NID, AVF). In terms of plant response, DDR, has been shown to activate salicylic acid accumulation associated with plant defense against biotroph pathogens.

总体而言，如果考虑光周期的生长，布朗产生更高浓度的已知具有遗传毒性的化合物（AVR，VERB和VERA），而其他三种分离株产生更多已知诱导细胞凋亡的化合物（NID，AVF）。就植物反应而言，DDR已被证明可激活与植物防御生物营养病原体相关的水杨酸积累。

Melanin and β-glucans. Melanins are complex and multi-functional polyphenol biopolymers that can act as antioxidants, confer resistance to radiation and osmotic stress, work as energy storage, and be involved in pathogenesis

黑色素和β-葡聚糖。黑色素是一种复杂的多功能多酚生物聚合物，可以作为抗氧化剂，抵抗辐射和渗透压，储存能量，并参与发病机制

. There are at least eight types of melanin

.黑色素至少有八种

. They can accumulate on various fungal structures and at different ratios playing different roles in pathogenicity

。它们可以积累在各种真菌结构上，并以不同的比例在致病性中发挥不同的作用

and can also confer some resistance to antifungal drugs

也可以赋予抗真菌药物一定的耐药性

. In our work, BROWN contained 1.48 mg melanin/g DW, 1.6- to 2.3-fold higher than the rest of the isolates, explaining the brown coloration; however, this isolate did not show significant advantage when exposed to four xenobiotic compounds, Supplementary Fig. 8. The CP isolates had between 0.4 and 1.6% DW melanin which is within the common range, 0.4 and 8.0% melanin, in fungal biomass.

在我们的工作中，棕色含有1.48毫克黑色素/克DW，比其他分离株高1.6至2.3倍，解释了棕色的着色；然而，当暴露于四种异生素化合物时，该分离物没有显示出显着的优势，补充图8。CP分离株的DW黑色素含量在0.4%至1.6%之间，在真菌生物量的常见范围内，黑色素含量为0.4%至8.0%。

, though we only extracted and quantified insoluble melanin

，尽管我们只提取和定量了不溶性黑色素

. In humans, the photoprotection effect of melanin in skin depends not only on the types of melanin present (eumelanin, pheomelanin) but also their ratios

在人类中，黑色素在皮肤中的光保护作用不仅取决于存在的黑色素类型（真黑色素，褐黑色素），还取决于它们的比例

. Thus, further work to characterize melanin in CP would be helpful. It was recently discovered that most enzymes in the 1,8-dihydroxynapthalene (1,8-DHN) melanin pathway are shared with the perylene quinone mycotoxin pathway, e.g. altertoxin (ATX)

因此，进一步研究CP中黑色素的特征将有所帮助。最近发现，1,8-二羟基萘（1,8-DHN）黑色素途径中的大多数酶与苝醌真菌毒素途径共享，例如交替毒素（ATX）

, thus, this possibility could be explored in CP to better understand its interaction with the peanut plant.

因此，可以在CP中探索这种可能性，以更好地了解其与花生植物的相互作用。

Two interesting observations derive from the quantification of β-[1,3] and β-[1,4] polysaccharides of chitin. First, that β-[1,3]-glucan-chitin make a particularly flexible viscoelastic network

两个有趣的观察结果来自几丁质的β-[1,3]和β-[1,4]多糖的定量。首先，β-[1,3]-葡聚糖几丁质形成了一个特别灵活的粘弹性网络

, therefore, the rubbery, flexible consistency of the red morphotype, RED, may be the result of its significantly higher content of β-glucans compared to the rest of the isolates. Second, that chitin and chitin-related compounds activate the biosynthesis of jasmonic acid (JA), which is associated with the plant response to necrotroph pathogens.

因此，与其他分离株相比，红色形态型红色的橡胶状柔性稠度可能是其β-葡聚糖含量显着更高的结果。其次，几丁质和几丁质相关化合物激活茉莉酸（JA）的生物合成，这与植物对坏死性病原体的反应有关。

Mating type. This is the first report of MAT1 locus genes and heterothallic nature of

配合类型。这是MAT1基因座基因和杂合性的首次报道

Cercosporidium personatum

人尾孢霉

. Here we compared three new CP genomes with those previously reported

在这里，我们将三个新的CP基因组与先前报道的基因组进行了比较

and show each harbored only one MAT1 idiomorph, either MAT1-1 or MAT1-2 but not both, therefore indicating these isolates are heterothallic. We had reported CP contig_298 contained MAT1 locus

并显示每个仅含有一个MAT1自形，MAT1-1或MAT1-2，但不是两者，因此表明这些分离株是异源的。我们报道了CP重叠群298含有MAT1基因座

. Here we detailed the structure of MAT1 locus in chromosome 12 flanked by DOT biosynthesis gene mini-clusters, the gene sequences of MAT1-1 and MAT1-2, and primers for their detection in population studies. Following the nomenclature for mating type genes in filamentous ascomycetes

在这里，我们详细介绍了12号染色体上MAT1基因座的结构，两侧是点生物合成基因微簇，MAT1-1和MAT1-2的基因序列，以及在人群研究中检测它们的引物。遵循丝状子囊菌交配型基因的命名法

, the four CP isolates studied here had the High Mobility Group (HMG) type, MAT1-2 gene (am_ctg298-0.26); whereas CBS 151,044

，这里研究的四个CP分离株具有高迁移率族（HMG）型MAT1-2基因（am\U ctg298-0.26）；鉴于CBS 151044

and IPAVE 0302

和IPAVE 0302

had the Alpha Box motif protein (MATα1) or MAT1-1, Supplementary Fig. 7. Equal ratios of mating types in fungal pathogen populations are indication of sexual reproduction and increased prospect of overcoming plant host resistance

具有α盒基序蛋白（MATα1）或MAT1-1，补充图7。真菌病原体种群中交配类型的相等比例表明有性繁殖，并增加了克服植物宿主抗性的前景

. Therefore, these primers will be crucial to analyze mating type distribution in the LLS pathogen populations.

因此，这些引物对于分析LLS病原体种群中的交配型分布至关重要。

Xenobiotics. This is the first report of response to fungicides by individual CP isolates using image analysis. Image analysis was recently developed to evaluate the recalcitrant growth habit of peanut leafspot pathogens CP and CA

异生素。这是使用图像分析的单个CP分离株对杀菌剂反应的首次报道。最近开发了图像分析来评估花生叶斑病菌CP和CA的顽固生长习性

. To test CP exposure to xenobiotic compounds we standardized inoculum size, introduced the use of PVDF membranes on the medium, and determined suitable incubation conditions that allowed results in 5 days. Normally, evaluating fungicide resistance in individual CP isolates would take 3–6 months, and the impracticality of this is pointed out in the literature.

为了测试CP暴露于异生素化合物，我们标准化了接种量，在培养基上引入了PVDF膜的使用，并确定了合适的孵育条件，可以在5天内获得结果。通常，评估单个CP分离株的杀菌剂抗性需要3-6个月，文献中指出了这一点的不切实际。

. A previous method developed to evaluate fungicide resistance used 15–20 peanut leafspot lesions from the field to obtain results in 14–30 days

先前开发的一种评估杀菌剂抗性的方法是从田间使用15-20个花生叶斑病变，以在14-30天内获得结果

by the detached leaf assay

通过离体叶片测定

. The method we describe is not only completed in 5 days, but it also allowed us to calculate the EC

。我们描述的方法不仅在5天内完成，而且还允许我们计算EC

of fungicides, whereas more accurate estimations can be obtained by increasing the number of isolates.

杀菌剂的数量，而通过增加分离株的数量可以获得更准确的估计。

Multiple applications of fungicides are necessary during the peanut cropping season to control leafspot diseases

在花生种植季节，需要多次施用杀菌剂来控制叶斑病

. Among these products are the sterol biosynthesis inhibitors (SBI) TEB and PROT, and the multi-site fungicide of the chloronitrile group CHLOR

这些产品包括甾醇生物合成抑制剂（SBI）TEB和PROT，以及氯腈类氯的多点杀菌剂

, the latter being used as the standard when comparing fungicides

，后者被用作比较杀菌剂的标准

. In our experiments, expression of eight glutathione S-transferase (GST) genes was highest in TAN and lowest in NRRL 64,463, which correlated with their levels of resistance to TEB and CHLOR (ea., 10 mg/L). GST catalyzes the conjugation of the glutathione sulfur atom to xenobiotic compounds resulting in detoxification.

在我们的实验中，八个谷胱甘肽S-转移酶（GST）基因的表达在TAN中最高，在NRRL 64463中最低，这与它们对TEB和CHLOR的抗性水平相关（ea。，10 mg/L）。GST催化谷胱甘肽硫原子与异生素化合物的结合，从而产生解毒作用。

. The three colored morphotype CP isolates, RED, TAN, BROWN, were collected in 2021, and had higher resistance to TEB, CHLOR, and PROT than NRRL 64,463 which was isolated in 2013, Supplementary Fig. 8. This is not surprising since the effectiveness of these fungicides has been reported to decrease over the years.

2021年收集了三种颜色的形态型CP分离株，红色，棕褐色，棕色，对TEB，CHLOR和PROT的抗性高于2013年分离的NRRL 64463，补充图8。这并不奇怪，因为据报道这些杀菌剂的有效性多年来有所下降。

. Caffeine was included in the tests because gene sm_ctg_390 − 0.41 which encodes for a caffeine resistance protein, was expressed 2,300-fold higher in TAN compared to the rest of the isolates, though no correlation was observed with its caffeine tolerance.

咖啡因被包括在测试中，因为编码咖啡因抗性蛋白的基因sm ctg U 390- 0.41在棕褐色中的表达比其他分离株高2300倍，尽管与其咖啡因耐受性没有相关性。

Ergosterol. Ergosterol (Ergosta-5,7,22-trien-3β-ol; C

麦角固醇。麦角甾醇（麦角甾醇-5,7,22-三烯-3β-醇；C

小时

O, MW:396.34) is an essential component and the most abundant sterol of fungal cell membranes and has an essential role in membrane stabilization

O、分子量：396.34）是真菌细胞膜的重要成分和最丰富的甾醇，在膜稳定中起着至关重要的作用

. Within fungal species, ergosterol content is considered rather constant

在真菌物种中，麦角固醇含量被认为是相当恒定的

whether a culture is grown in photoperiod or darkness

培养物是在光周期还是黑暗中生长

, hence it is often used to estimate fungal biomass in ecosystems

因此，它通常用于估计生态系统中的真菌生物量

. Thus, it was surprising to find that CP isolates grown in the same conditions had statistically significant differences in ergosterol content (

因此，令人惊讶的是，发现在相同条件下生长的CP分离株在麦角固醇含量上具有统计学上的显着差异(

< 0.01). RED grown in darkness reached 4.5 mg/g DW ergosterol, similar concentrations have been reported in other fungal pathogens, e.g., between 4 and 6 mg/g DW in

0.01）。在黑暗中生长的红色达到4.5 mg/g DW麦角固醇，其他真菌病原体的浓度也相似，例如，在4至6 mg/g DW之间

Trichoderma harzianum

哈茨木霉

, 3.9 mg/g DW in

，3.9毫克/克DW英寸

Botrytis cinerea

灰葡萄孢

and 6.7 mg/g DW in

和6.7毫克/克DW

Fusarium sporotrichioides

孢子丝镰刀菌

. In RED and TAN, the highest ergosterol content in photoperiod (

在红色和棕褐色中，光周期中麦角固醇含量最高(

< 0.01) (Fig.

<0.01）（图。

), correlated with high ergosterol biosynthesis gene expression, mevalonate kinase, diphospho-mevalonate kinase, and putative squalene monooxygenases (Fig.

)，与高麦角固醇生物合成基因表达，甲羟戊酸激酶，二磷酸甲羟戊酸激酶和假定的角鲨烯单加氧酶相关（图）。

), and high resistance to TEB (10 mg/L), Supplementary Fig. 8. These two isolates also showed a moderate ~ 5-fold increase in ergosterol content when grown in darkness, whereas BROWN and NRRL 64,463 experience 18-fold ergosterol increase in darkness, Fig.

)，以及对TEB的高抗性（10 mg/L），补充图8。这两个分离株在黑暗中生长时，麦角固醇含量也适度增加了5倍，而棕色和NRRL 64463在黑暗中麦角固醇含量增加了18倍。

. The ergosterol molecule and its biosynthesis genes are targets of important groups of fungicides such as azoles and polyenes, noting that changes in ergosterol abundance affect the level of susceptibility to these drugs

麦角固醇分子及其生物合成基因是唑类和多烯类等重要杀菌剂的靶标，注意到麦角固醇丰度的变化会影响对这些药物的敏感性水平

. Consequently, the sizable variation in ergosterol content among CP isolates and growing conditions, deserves consideration when assessing fungicide resistance in the LLS pathogen.

因此，在评估LLS病原体的杀菌剂抗性时，CP分离株和生长条件之间麦角固醇含量的巨大差异值得考虑。

Ergosterol is not produced in plants, therefore it is recognized as a “non-self” molecule by the plant immune system

麦角固醇不是植物产生的，因此它被植物免疫系统认为是一种“非自身”分子

. Ergosterol is an effector of the microbe/pathogen-associated molecular pattern (MAMP/PAMP) recognition along with chitin (β-glucans)

麦角固醇是微生物/病原体相关分子模式（MAMP/PAMP）识别以及几丁质（β-葡聚糖）的效应物

. Based on fresh weight, some CP morphotypes accumulated 0.5–1 mM ergosterol. It is known that as small as nanomolar concentrations of ergosterol can activate production of salicylic acid (SA) (response to biotrophs)

根据鲜重，一些CP形态型积累了0.5-1 mM麦角固醇。众所周知，只有纳摩尔浓度的麦角固醇才能激活水杨酸（SA）的产生（对生物营养的反应）

. However, the response to ergosterol also depends on the abundance of squalene, precursor of ergosterol, and on the ergosterol/squalene ratio where the transcription factor, WRKY40, has been shown to positively modulate JA and inactivate SA

然而，对麦角固醇的反应还取决于麦角固醇前体角鲨烯的丰度，以及麦角固醇/角鲨烯的比例，其中转录因子WRKY40已被证明可以正向调节JA并使SA失活

. Other CP morphotypes produced significantly higher concentration of genotoxic AQ. In general, genotoxicity, DNA damage, is known to activate SA production

其他CP形态型产生的遗传毒性AQ浓度明显更高。一般来说，已知遗传毒性，DNA损伤会激活SA的产生

. Yet, another morphotype accumulated up to 40-fold more chitin than other isolates. In plants, chitin activates the jasmonic acid biosynthesis pathway (response to nectrotrophs)

然而，另一种形态类型积累的几丁质比其他分离株多40倍。在植物中，几丁质激活茉莉酸的生物合成途径（对蜜环菌的反应）

. In summary, different CP morphotypes could facilitate suppression of either JA or SA. Such changes in JA or SA response could render a plant more susceptible to pathogens (here morphotypes) that have a different mode of invasion

总之，不同的CP形态型可以促进JA或SA的抑制。JA或SA反应的这种变化可能使植物更容易受到具有不同入侵模式的病原体（此处为形态型）的影响

. We speculate, CP morphotypes could cooperate among themselves to achieve plant host invasion.

我们推测，CP形态类型可以相互合作以实现植物宿主入侵。

Methods

方法

Fungal isolation

真菌分离

Color variant isolates of

颜色变异分离株

Cercosporidium personatum

人尾孢霉

(CP) used in this work were initiated in culture from sporulating late leafspot lesions of peanut plants cv. Georgia-06G, collected in October 2021 at the University of Georgia Coastal Plain Experiment Station, Lang Farm in Tifton, Georgia (Lat.: 31.476099 N; Long.: -83.5248768 W). Lesions were dried at room temperature and stored in glass vials for six months at 4 °C.

这项工作中使用的（CP）是从2021年10月在佐治亚州提夫顿朗农场佐治亚大学海岸平原实验站收集的花生植物品种Georgia-06G的孢子形成晚期叶斑病变的培养物中开始的（纬度：31.476099 N；长：83.5248768 W）。病变在室温下干燥，并在4°C下保存在玻璃瓶中六个月。

After five months of incubation, tissues with the original brown morphology, having dark brown, pulvinate stroma with a hard texture (NPO22: BROWN), and those of a tan color morphological variant having similar stroma covered with tan or light brown prostrate hyphae (NPT22: TAN), were excised from the same plate and sub-cultured.

孵育五个月后，具有原始棕色形态的组织，具有深棕色，具有硬纹理的脉状基质（NPO22：棕色），以及具有类似基质的棕褐色形态变体的组织，所述基质覆盖有棕褐色或浅棕色匍匐菌丝（NPT22：棕褐色），从同一平板上切下并传代培养。

A red variant, having red stroma with a smooth and soft texture (NPR22: RED) was obtained from a separate culture. The morphology of BROWN, TAN, RED cultures was characterized. Excised stromatal tissues of the cultures were homogenized separately in 1 ml sterile water using a LabGEN 125 Tissue Homogenizer (Cole Parmer, Vernon Hills, IL) and transferred to fresh potato-dextrose agar (PDA) plates (potato dextrose broth Difco cat.

从单独的培养物中获得具有光滑柔软纹理的红色基质的红色变体（NPR22：红色）。表征了棕色，棕褐色，红色培养物的形态。使用LabGEN 125组织匀浆器（Cole Parmer，Vernon Hills，IL）将切下的培养物基质组织分别在1 ml无菌水中匀浆，并转移至新鲜马铃薯葡萄糖琼脂（PDA）平板（马铃薯葡萄糖肉汤Difco cat）。

# 254920, with 15 g/L agar Fisher cat. # BP1423), to cultivate morphotype cultures and initiate nonstromatic growth, e.g. filamentous hyphae or conidia, for further study. All these isolates were vouchered with the Valdosta State University (VSC) Fungarium, uploaded to MycoPortal, and submitted to the USDA-NRRL culture collection in Peoria, IL, USA..

#254920，含15克/升琼脂Fisher猫BP1423），以培养形态型培养物并启动非色素生长，例如丝状菌丝或分生孢子，以供进一步研究。所有这些分离株均由瓦尔多斯塔州立大学（VSC）真菌库提供，上传至MycoPortal，并提交给美国伊利诺伊州皮奥里亚的USDA-NRRL培养物保藏中心。。

Morphological characterization

形态学表征

Morphological observations were conducted for three isolates of the original brown morphotype, three of the red morphotype, and one tan form. Following the transfer of conidia or filamentous hyphae induced by homogenization to fresh PDA plates, 9-day old cultures were examined at 400X magnification for the presence or absence of conidia, verrucose hyphae, and orange color extracellular pigments present either as droplets near hyphae or as crystallizations on hyphal surfaces.

对三个原始棕色形态型分离株，三个红色形态型分离株和一个棕褐色分离株进行形态学观察。将匀浆诱导的分生孢子或丝状菌丝转移到新鲜的PDA平板上后，在400倍放大倍数下检查9天大的培养物是否存在分生孢子，疣状菌丝和橙色细胞外色素，这些色素以菌丝附近的液滴或菌丝表面的结晶形式存在。

Five fields of view chosen at random were observed for each sample. Scanning electron microscopy (SEM) was conducted using a JEOL 6480 LV scanning electron microscope (JEOL, Tokyo, Japan) to confirm and characterize verrucose hyphae. Tissues for SEM analyses were prepared from sections of 9-day old cultures that had been dried at 46 °C for 24 h.

对每个样品观察随机选择的五个视野。使用JEOL 6480 LV扫描电子显微镜（JEOL，东京，日本）进行扫描电子显微镜（SEM）以确认和表征疣状菌丝。用于SEM分析的组织是从9天大的培养物切片中制备的，该培养物已在46°C下干燥24小时。

Tissues were affixed to aluminum SEM pegs using double-sided carbon tape and coated with gold/palladium using a Denton Vacuum Desk IV sputter-coater (Moorestown, New Jersey, USA). The presence or absence of verrucose hyphae were observed at 1000X to 3000X. When present, verrucose lobe densities were rated as low, medium, or high, and the diameters of verrucose hyphae and individual lobes were measured..

使用双面碳带将组织固定在铝SEM钉上，并使用Denton真空台式IV溅射镀膜机（Moorestown，New Jersey，USA）用金/钯涂覆。在1000倍至3000倍时观察到疣状菌丝的存在或不存在。当存在时，疣状叶密度被评定为低，中或高，并测量疣状菌丝和单个叶的直径。。

DNA extraction and sequencing

DNA提取和测序

The three color-CP isolates, BROWN, RED and TAN, were grown on PDA medium at 26 °C as initial inoculum. Approximately 1 cm

棕色，红色和棕褐色三种颜色的CP分离株在26℃的PDA培养基上生长作为初始接种物。大约1厘米

of each culture was separately placed in sterile test tubes containing 2 mL sterile distilled water, ground with a LabGEN 125 Tissue Homogenizer, then, 400 µL of each culture suspension were spread on PDA plates. The plates were incubated at 26 °C ± 0.5 °C 16 h light/8 h dark to induce sporulation.

将每种培养物的培养物分别置于含有2 mL无菌蒸馏水的无菌试管中，用LabGEN 125组织匀浆器研磨，然后将400µL每种培养液悬浮在PDA平板上。将板在26℃±0.5℃下孵育16小时光照/8小时黑暗以诱导孢子形成。

Biomass, including spores and hyphae were harvested after 9, 13 and 18 days for BROWN, RED and TAN, respectively. DNA was extracted from the fungal biomass using DNeasy Plant Mini Kit in a QIAcube robot (both from Qiagen, Redwood City, CA, USA). Genomic DNA libraries were prepared for each color CP isolate using Illumina TruSeq Prep Kit V2 (Illumina, San Diego, CA), and sequenced as paired end (PE) 150 base pairs (bp) on Illumina NovaSeq 6000 System (Illumina, San Diego, CA) at the LC Sciences, Houston, TX..

分别在棕色，红色和棕褐色的9,13和18天后收获生物量，包括孢子和菌丝。使用QIAcube机器人（均来自美国加利福尼亚州红木市的Qiagen）中的DNeasy Plant Mini Kit从真菌生物质中提取DNA。使用Illumina TruSeq Prep Kit V2（Illumina，圣地亚哥，加利福尼亚）为每种颜色的CP分离物制备基因组DNA文库，并在德克萨斯州休斯顿的LC Sciences的Illumina NovaSeq 6000系统（Illumina，圣地亚哥，加利福尼亚）上测序为配对末端（PE）150个碱基对（bp）。。

Mapping and analysis of genomic variants

基因组变异的定位和分析

Given the various morphologies of the colored CP isolates, we first confirmed their taxonomic identification by mapping the sequencing reads to highly conserved genes commonly used in phylogenetics of Ascomycetes. After removing low quality reads and potential adapters, reads were mapped to 8511 bp ribosomal-RNA operon, 5523 base pairs (bp) of the RNA-polymerase II largest subunit (RPB1) and to 4434 bp of the RNA-polymerase II second largest subunit (RPB2) of the reference genome NRRL 64,463.

鉴于有色CP分离株的各种形态，我们首先通过将测序读数映射到子囊菌系统发育中常用的高度保守基因来确认它们的分类学鉴定。去除低质量读物和潜在的衔接子后，读物被定位到8511 bp的核糖体RNA操纵子，5523个碱基对（bp）的RNA聚合酶II最大亚基（RPB1）和4434 bp的RNA聚合酶II第二大亚基（RPB2）参考基因组NRRL 64463。

Reads of RED, BROWN, and TAN (this work) and the reads of NRRL 64,463 (NCBI: SRR22033838) genome.

红色，棕色和棕褐色的读数（这项工作）和NRRL 64463（NCBI:SRR22033838）基因组的读数。

were then mapped to the 1061 contigs of NRRL 64,463, and genetic variants searched using the following parameters: ploidy = 1, minimum count = 2, minimum coverage = 10 (10% of the read coverage at that locus), minimum frequency = 35%. Genome-wide variants (insertion/deletions (InDels)), and single- or multi-nucleotide polymorphisms (SNPs, MNPs) were recorded.

然后将其定位到NRRL 64463的1061个重叠群，并使用以下参数搜索遗传变异：倍性 = 1, 最小计数=2，最小覆盖率为10（该基因座读取覆盖率的10%），最小频率为35%。记录了全基因组变异（插入/缺失（InDels））和单核苷酸或多核苷酸多态性（SNP，MNP）。

Variants were detected using the same parameters and a minimum frequency of 1% cutoff was used to observe allele frequency distributions, and only hyper-allelic variants were computed. All analyses were performed using CLC Genomics Workbench 23.0.4 (Qiagen, Aarhus, Denmark)..

使用相同的参数检测变体，并使用1%截止值的最小频率观察等位基因频率分布，并且仅计算超等位基因变体。所有分析均使用CLC Genomics Workbench 23.0.4（Qiagen，奥胡斯，丹麦）进行。。

RNA sequencing and data analysis

RNA测序和数据分析

The three color-CP isolates and the reference culture NRRL 64,463 were grown each on 20 plates of PDA medium and incubated at 26 °C ± 0.5 °C with photoperiod 16 h light/8 h dark for 12 days. Their biomasses were collected on separate 50 mL sterile centrifuge tubes and stored at – 80 °C approximately 2–3 weeks until processing.

将三色CP分离株和参考培养物NRRL 64463分别在20个PDA培养基平板上生长，并在26℃±0.5℃下用光周期16小时光照/8小时黑暗孵育12天。将它们的生物量收集在单独的50 mL无菌离心管中，并在–80°C下保存约2–3周，直到处理。

Total RNA was extracted from two biological replicates of each isolate, mRNA libraries were prepared using TruSeq RNA library prep kit v2 (Illumina, San Diego, CA), and sequenced on Illumina NovaSeq 6000 at the University of Minnesota Genomics Center, MN, (.

从每个分离物的两个生物学重复中提取总RNA，使用TruSeq RNA文库制备试剂盒v2（Illumina，圣地亚哥，加利福尼亚）制备mRNA文库，并在明尼苏达大学基因组学中心的Illumina NovaSeq 6000上测序。

https://genomics.umn.edu

). RNA sequencing data were normalized using trimmed mean of million (M) values (TMM) method

)。使用百万（M）值的修剪平均值（TMM）方法对RNA测序数据进行归一化

, and Principal Component Analysis (PCA)

，以及主成分分析（PCA）

was performed to visualize the variation between isolates. Based on the annotated reference genome NRRL 64,463, differential expression analysis was performed according to Mortazavi et al.

进行可视化分离株之间的变化。基于注释的参考基因组NRRL 64463，根据Mortazavi等人的方法进行差异表达分析。

with parameters set to Bonferroni corrected p-value ≤ 1.0E-5, and Log

参数设置为Bonferroni校正的p值≤ 1.0E-5，并记录

of absolute fold change ≥ 2. Heat maps were created for various groups of genes of interest including pathogenicity/virulence genes identified by pattern-hit initiated BLAST (PHI-BLAST)

绝对倍数变化≥2。为各组感兴趣的基因创建了热图，包括通过模式命中引发的BLAST（PHI-BLAST）鉴定的致病性/毒力基因

, carbohydrate active enzymes (dbCAN2)

，碳水化合物活性酶（dbCAN2）

, apoplastic and cytoplasmic effectors according to Effector_P

，根据Effector\u P的质外和细胞质效应子

, and dothistromin/melanin/chitin/ergosterol biosynthesis related genes

，以及dothistromin/黑色素/几丁质/麦角固醇生物合成相关基因

. In addition, using previously identified secondary metabolite gene clusters

此外，使用先前确定的次级代谢产物基因簇

, we searched the genomes of color-CP isolates for predicted potential amino acid changes. All analyses were performed using CLC Genomics Workbench 23.0.2.

，我们搜索了彩色CP分离株的基因组，以预测潜在的氨基酸变化。所有分析均使用CLC Genomics Workbench 23.0.2进行。

Quantitative real-time-PCR of pigment biosynthesis genes

色素生物合成基因的实时定量PCR

Using the genome annotation of NRRL 64,463

使用NRRL 64463的基因组注释

, both genes and transcripts of five genes in the dothistromin-biosynthesis pathway (

，dothistromin生物合成途径中五个基因的基因和转录本(

pksA

vbsA

dotA

, verb,

，动词，

avfA

) and two in the melanin-biosynthesis pathway (

)黑色素生物合成途径中有两个(

pks1

t4hnr

) were selected for quantitative Real-Time PCR (qPCR). Genes and transcripts were aligned and primers were designed to span introns using Clone Manager v.11 (Sci. Ed. Software LLC, Westminster, CO). The three color-CP isolates and the reference NRRL 64,463 were grown on multiple PDA medium plates and incubated at 26 °C ± 0.5 °C with photoperiod 16 h light/8 h dark for 12 days.

)选择用于定量实时PCR（qPCR）。使用Clone Manager v.11（Sci.Ed.Software LLC，Westminster，CO）对基因和转录本进行比对，并设计引物以跨越内含子。将三色CP分离株和参考NRRL 64463在多个PDA培养基平板上生长，并在26℃±0.5℃下用光周期16小时光照/8小时黑暗孵育12天。

Fungal biomass was collected on separate 50 mL sterile centrifuge tubes, stored at -80 °C, and total RNA was extracted within one week after collection. Five cDNA reaction synthesis were prepared using 8 µg of total RNA from each of three biological replicates with a combination of random hexamers and oligo-dT.

在单独的50 mL无菌离心管上收集真菌生物量，保存在-80°C，收集后一周内提取总RNA。使用随机六聚体和oligo-dT的组合，使用来自三个生物学重复中的每一个的8µg总RNA制备了五个cDNA反应合成。

Superscript III First Strand Synthesis Super Mix (Cat. # 18080400) (Invitrogen by Thermo Fisher Scientific, Waltham, MA) was used according to manufacturer’s instructions. qPCR was performed on an Applied Biosystems QuantStudio 7 Pro instrument (Thermo Fisher Scientific, Waltham, MA), using RT2-SYBR Green ROX qPCR Mastermix (Cat.

根据制造商的说明使用Superscript III First Strand Synthesis Super Mix（目录号#18080400）（Invitrogen，Thermo Fisher Scientific，Waltham，MA）。qPCR在Applied Biosystems QuantStudio 7 Pro仪器（Thermo Fisher Scientific，Waltham，MA）上使用RT2-SYBR Green ROX qPCR Mastermix（Cat。

# 330523) (QIAGEN, Germantown, MD). The qPCR was performed in 24 µL reactions with 5 µL of a ¼ dilution of cDNA, 0.4 µM of each primer and 12 µL qPCR Mastermix. Conditions for amplification were: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 58 °C, and dissociation curve analysis of 15 s at 95 °C, 1 min at 58 °C, 15 s at 95 °C.

#330523）（QIAGEN，Germantown，MD）。qPCR在24µL反应中进行，其中5µL¼稀释的cDNA，0.4µM每种引物和12µL qPCR Mastermix。扩增条件为：在50°C下2分钟，在95°C下10分钟，然后在95°C下15 s，在58°C下1分钟的40个循环，以及在95°C下15 s的解离曲线分析，在58°C下1分钟，在95°C下15秒。

Each biological sample was analyzed for eight primer sets and for a housekeeping gene (Actin), using three biological samples per treatment and three technical replicates per biological sample. Data were analyzed using Design & Analysis 2.6.0 software provided with the instrument QuantStudio 7 Pro (Thermo Fisher Scientific, Waltham, MA).

每个生物样品分析八个引物组和管家基因（肌动蛋白），每个处理使用三个生物样品，每个生物样品使用三个技术重复。使用仪器QuantStudio 7 Pro（Thermo Fisher Scientific，Waltham，MA）提供的Design＆Analysis 2.6.0软件分析数据。

The list of primers used for qPCR is shown in Supplementary Table 5..

用于qPCR的引物列表见补充表5。。

Melanin quantification

黑色素定量

To determine melanin production, RED, TAN, BROWN and NRRL 64,463 CP isolates were grown on PDA medium incubated at 26 °C ± 0.5 °C in the dark for 17 days. Biomass of two plates per culture of each isolate were placed into separate 50 mL centrifuge tubes and stored at -80 °C until processing. The samples were lyophilized at 3 Pa for 16 h, biomass weighted with a 5-decimal place accuracy in a XPR105DR, XPR Analytical balance (Cat.

为了确定黑色素的产生，将红色，棕褐色，棕色和NRRL 64463 CP分离株在PDA培养基上生长，该培养基在26℃±0.5℃下在黑暗中孵育17天。将每个分离物的每个培养物的两个平板的生物量放入单独的50毫升离心管中，并在-80℃下储存直至处理。将样品在3 Pa下冻干16小时，在XPR105DR，XPR分析天平（Cat。

# 30355342, Mettler Toledo, Columbus, OH), and processed for the removal of AQ that interfere with the quantification of melanin as follows: the tubes were loaded with 16 zirconium beads (12 of 2.8 mm dia. and 4 of 6.5 mm dia.) and 15 mL of ethyl acetate, sealed with matching caps and the fungal mass was pulverized in an OMNI Bead Ruptor (OMNI International, cat.

#30355342，Mettler Toledo，Columbus，OH），并处理以去除干扰黑色素定量的AQ，如下所示：管中装有16个锆珠（12个直径为2.8毫米，4个直径为6.5毫米）和15毫升乙酸乙酯，用匹配的盖子密封，真菌块在OMNI珠破裂器（OMNI International，cat）中粉碎。

# 19-042E, Kennesaw, GA) at 5.5 m/sec for 45 s. The slurry obtained was centrifuged at 1,800 × .

#19-042E，肯尼索，佐治亚州）以5.5米/秒的速度持续45秒。将获得的浆料以1800倍离心。

克

for 8 min in a Model 6755 LSE compact centrifuge (Corning Life Sciences); the supernatant was carefully removed with a glass pipette. Extraction of AQ was repeated three more times with 15 mL ethyl acetate followed by centrifugation and removal of the supernatant. The precipitate was allowed to dry in a fume hood at ambient temperature for 3 h.

在6755型LSE紧凑型离心机（康宁生命科学）中放置8分钟；用玻璃移液管小心地除去上清液。用15 mL乙酸乙酯重复提取AQ三次，然后离心并除去上清液。使沉淀物在通风橱中在环境温度下干燥3小时。

After removal of AQ, melanin was extracted according to reported protocols.

去除AQ后，根据报道的方案提取黑色素。

, with slight modifications as follows: the finely ground biomass (100 mg/sample) was placed in 25 mL Erlenmeyer flasks, 20 mL of freshly prepared 0.5 M NaOH were added, each flask was sealed with three layers of parafilm, and incubated overnight in a shaker incubator at 25 °C and 150 rpm. The extract was then centrifuged at 7,200 .

，稍作修改如下：将精细研磨的生物质（100 mg/样品）置于25 mL锥形瓶中，加入20 mL新鲜制备的0.5 M NaOH，每个烧瓶用三层parafilm密封，并在25°C和150 rpm的摇床培养箱中孵育过夜。然后将提取物在7200离心。

克

and the supernatant was vacuum filtered using a glass fiber filter type E (Gelman Instrument Company, Ann Arbor, MI) as prefilter and a 0.22 μm PVDF membrane. The filtrate was placed in 50 mL polypropylene centrifuge tubes with a small stirrer, added 1 N HCl until reaching pH: 1.5, and allowed to rest for one hour at ambient temperature.

并使用E型玻璃纤维过滤器（Gelman Instrument Company，Ann Arbor，MI）作为预过滤器和0.22μm PVDF膜对上清液进行真空过滤。将滤液用小搅拌器置于50 mL聚丙烯离心管中，加入1 N HCl直至pH值达到1.5，并在环境温度下静置1小时。

Then, the tubes were centrifuged at 7,200 .

然后，将管在7200离心。

克

and 15 °C for 70 min to precipitate the melanin. The pellet was washed once with 3 mL 0.1 N HCl, centrifuged again at 7,200

并在15°C下放置70分钟以沉淀黑色素。将沉淀用3 mL 0.1 N HCl洗涤一次，然后在7200下再次离心

克

for 15 min, then resuspended first in 300 µL 0.1 N HCl that were transferred to a pre-weighed 4 mL glass vial, another 200 µL of 0.1 N HCl were used to collect the remainder and added to the glass vial. Vials containing 500 µL of melanin suspension were frozen at -80 °C for 10 min and lyophilized. The final weight of the precipitate was measured in a 5-decimal place balance XPR105DR.

持续15分钟，然后首先重悬于300µL 0.1 N HCl中，将其转移到预先称重的4 mL玻璃瓶中，再使用200µL 0.1 N HCl收集剩余物并添加到玻璃瓶中。将含有500μL黑色素悬浮液的小瓶在-80℃冷冻10分钟并冻干。沉淀物的最终重量在小数点后5位的天平XPR105DR中测量。

Results were expressed as mg of melanin per g of dry weight of fungal biomass. Statistical analysis of the results was performed as mean comparison by Tukey’s test, using SYSTAT (Sigma Plot v. 15)..

结果表示为每克真菌生物量干重的黑色素毫克数。使用SYSTAT（Sigma Plot v。15），通过Tukey检验对结果进行统计分析，作为平均值比较。。

Presence of chitin, or 1–3 and 1-4-β-glucans

存在几丁质或1-3和1-4-β-葡聚糖

Standard β-glucan (CAS 9012-72-0, Prod. No. 346210) was purchased from Millipore (Sigma-Aldrich, St. Louis, MO). Two mg of β-glucan were dissolved in 100 µL sterile distilled water. Twenty µL of that solution were placed in 380 µL sterile distilled water, added 2 µL of 5 mM calcofluor white (Cat. # 29067, Biotium Inc., Fremont, CA), wrapped in aluminum foil and incubated for 20 min at ambient temperature ~ 22 °C.

标准β-葡聚糖（CAS号9012-72-0，产品号346210）购自Millipore（密苏里州圣路易斯的Sigma-Aldrich）。将两mgβ-葡聚糖溶解在100µL无菌蒸馏水中。将20µL该溶液置于380µL无菌蒸馏水中，加入2µL 5 mM calcofluor white（目录号29067，Biotium Inc.，Fremont，CA），用铝箔包裹，并在环境温度22°C下孵育20分钟。

Four technical replicates of 50 µL of the calcofluor-treated solution were applied to a 96-well black ultra-thin flat clear bottom plate (Corning; Cat. # 3615, Corning, NY) containing 50 µL per well of sterile distilled water and proceeded to make two-fold dilutions of the sample. The plate was read in a Synergy HTX multi-mode reader, BioTek (Agilent, Santa Clara, CA), using a tungsten light excitation filter 360/40 nm, emission filter 460/40 nm and 10 reads per data point, using Gen 5 Software 2.09.1.

将50μlCalcofluor处理过的溶液的四个技术重复应用于96孔黑色超薄透明平板（Corning；目录号#3615，Corning，NY），每孔含有50μL无菌蒸馏水，并进行样品的两倍稀释。使用Gen 5软件2.09.1，使用钨激发滤光片360/40 nm，发射滤光片460/40 nm和每个数据点10次读数，在Synergy HTX多模式读取器BioTek（加利福尼亚州圣克拉拉，安捷伦）中读取该板。

Reads of the first 6 dilutions followed a linear relationship and the fluorescence values were used as reference for calculations. The four CP isolates were grown for 12 days on PVDF membranes at 25 °C in the dark, then the biomass was harvested, placed into 50 mL centrifuge tubes at -80 °C overnight before lyophilizing.

前6次稀释的读数遵循线性关系，荧光值用作计算参考。将四种CP分离株在25℃的PVDF膜上在黑暗中生长12天，然后收获生物质，在-80℃下放置在50毫升离心管中过夜，然后冻干。

From each culture, three biological replicates of 20 mg dry biomass were placed into 2 mL OMNI tubes containing 3 × 2.8 mm zirconium beads. Sterile water, 400 µL, was added to each tube and the biomass was homogenized in an OMNI Bead Ruptor at 5 m/s for 30 s three times with 1 min rest between cycles.

从每种培养物中，将三个20 mg干生物质的生物复制品放入含有3×2.8 mm锆珠的2 mL OMNI管中。向每个试管中加入400µL无菌水，并将生物质在OMNI珠破裂器中以5 m/s的速度匀浆30 s三次，循环之间休息1分钟。

Two µL of 5 mM calcofluor white were added to each tube, mixed, covered with aluminum foil, and incubated at room temperature ~ 22 °C for 20 min. After incubation, the tubes were centrifuged for 5 min at 20,800 .

向每个管中加入两µL 5 mM calcofluor white，混合，用铝箔覆盖，并在室温〜22°C下孵育20分钟。温育后，将管在20800离心5分钟。

克

to precipitate fungal biomass, then 380 µL of supernatant were removed to reduce background fluorescence and replaced with 380 µL sterile distilled water. A total of 50 µL of sterile distilled water were added to the wells of a 96-well black ultra-thin flat clear bottom plate. Three technical replicates per biological sample were prepared by adding 50 µL of culture homogenate treated with calcofluor, and consecutive two-fold dilutions were prepared on the 12 well-columns of each plate.

为了沉淀真菌生物量，然后除去380µL上清液以减少背景荧光，并用380µL无菌蒸馏水代替。将总共50µL无菌蒸馏水添加到96孔黑色超薄透明平板的孔中。通过添加50µL用calcofluor处理的培养匀浆，每个生物样品制备三个技术重复，并在每个平板的12孔柱上制备连续的两倍稀释液。

A tube without fungal biomass was processed with calcofluor in the same manner and used as control. Reads from the control wells at each dilution were subtracted from the reads of each sample at the corresponding dilutions to account for fluorescence background. Slopes and intercepts of the Log.

用calcofluor以相同的方式处理没有真菌生物质的管并用作对照。从每个样品在相应稀释度下的读数中减去每个稀释度下对照孔的读数，以说明荧光背景。原木的坡度和截距。

transformed fluorescence curves for the first six dilutions, as well as maximum fluorescence on the first dilution were subjected to analysis of variance (ANOVA) and mean comparisons by Tukey’s test using SYSTAT (Sigma Plot V. 15).

使用SYSTAT（Sigma Plot V。15）通过Tukey检验对前六次稀释的转化荧光曲线以及第一次稀释的最大荧光进行方差分析（ANOVA）和平均比较。

Pathogenicity

致病性

Twelve-day old cultures of the three color-CP isolates and NRRL 64,463 were grown on PDAP medium, that is PDA medium supplemented with 25 mL/L of peanut-leaf extract (PDAP). The leaf extract was prepared by blending 300 g Georgia 06G leaves in 500 mL distilled water, filter sterilized through 0.22 μm and stored at -20 °C until use.

三色CP分离株和NRRL 64463的12日龄培养物在PDAP培养基上生长，PDAP培养基是补充有25 mL/L花生叶提取物（PDAP）的PDA培养基。通过将300克乔治亚06G叶子在500毫升蒸馏水中混合，通过0.22μm过滤灭菌并在-20℃下储存直至使用来制备叶子提取物。

The leaf extract was added after cooling the autoclaved PDA medium to 55 °C, thoroughly mixed and then poured into Petri dishes. Six leaves, second and third from the top of grow-light illuminated indoor-grown peanut cultivars Georgia-06G and AU-NPL17, were used in detached-leaf pathogenicity assays.

在将高压灭菌的PDA培养基冷却至55℃后加入叶提取物，充分混合，然后倒入培养皿中。六片叶子，从生长光照射的室内生长的花生品种Georgia-06G和AU-NPL17顶部的第二片和第三片叶子，用于离体叶片致病性测定。

. A total of 24 inoculation sites were used such that four leaflets of six leaves per cultivar received 3 µL of inoculum. Observations were performed every other day, starting 5 days after inoculation, though symptoms were confirmed after 2–3 weeks, and the experiment was repeated.

。总共使用了24个接种位点，使得每个品种六片叶子的四个小叶接受3μL接种物。从接种后5天开始，每隔一天进行一次观察，尽管在2-3周后症状得到确认，并重复实验。

Chemical analysis

化学分析

Reagents and materials

试剂和材料

HPLC-grade organic solvents used for fungal mass extraction and for preparation of the mobile phase were purchased from Fisher (Suwanee, GA). HPLC-grade water was prepared in the laboratory with a ZD20 four-bowl Milli-Q water system (Millipore). Formic acid (LS/MS, Optima) was purchased from Fisher Scientific (Cat.

用于真菌质量提取和制备流动相的HPLC级有机溶剂购自Fisher（Suwanee，GA）。在实验室中用ZD20四碗Milli-Q水系统（Millipore）制备HPLC级水。甲酸（LS/MS，Optima）购自Fisher Scientific（Cat。

# A117-50). Aliquots of anthraquinones, nidurufin, averantin, averufin, averufanin, and aversin were a generous gift from Dr. Kimiko Yabe (Fukui University of Technology, Japan). Versicolorin B, versicolorin A, norsolorinic acid, versiconol, versiconol acetae, O-methylsterigmatocystin, sterigmatocystin, dihydro sterigmatocystin, dihydro O-methylsterigmatocystin, dihydroxyaflavinine, asparason A, aflatrem, emodin, aflatoxins B.

#A117-50）。蒽醌，nidurufin，averantin，averufin，averufanin和aversin的等分试样是Kimiko Yabe博士（日本福井理工大学）的慷慨礼物。Versicolorin B，Versicolorin A，norsolorinic acid，versiconol，versiconol acetate，O-甲基杂色曲霉素，杂色曲霉素，二氢杂色曲霉素，二氢O-甲基杂色曲霉素，二羟基黄曲霉素，天冬氨酸A，黄曲霉素，大黄素，黄曲霉毒素B。

, B

，B

, G

，克

, G

，克

, M

，米

, and B

，和B

2a级

were made available as a part of the fungal secondary metabolites collection at the NPRL. The chromatographic purity of any of the listed above compounds exceeded 92%. Ergosterol (CAS_No. 57-87-4, Prod No. PHR1512) and squalene (CAS No. 111-02-4, Prod No. S3626, ~ 98%) were purchased from Millipore (Sigma-Aldrich Inc., St.

作为NPRL真菌次级代谢产物收集的一部分。上述任何化合物的色谱纯度均超过92%。麦角固醇（CAS号57-87-4，产品号PHR1512）和角鲨烯（CAS号111-02-4，产品号S3626，〜98%）购自Millipore（Sigma-Aldrich Inc.，St。

Louis, MO)..

密苏里州路易斯市）。。

Sample preparation

样品制备

Five biological replicates for each of the three colored CP isolates, RED, TAN, BROWN and NRRL 64,463 grown at 25 °C for 12 days with 16/8 h photoperiod, and another five replicates grown in darkness, were used for chemical profiling of secondary metabolites/pigments. After incubation, the fungal biomass was removed from each of the disks with a spatula and separately placed into 50-mL polypropylene centrifugal tubes (Corning 430290, Millipore-Sigma CLS430290) followed by lyophilization at 3 kPa for 16 h.

三种颜色的CP分离株（红色，棕褐色，棕色和NRRL 64463）在25°C下生长12天，光周期为16/8小时，每个分离株有五个生物学重复，另外五个重复在黑暗中生长，用于次级代谢产物/色素的化学分析。温育后，用抹刀从每个圆盘中取出真菌生物量，并分别放入50 mL聚丙烯离心管（Corning 430290，Millipore Sigma CLS430290）中，然后在3 kPa下冻干16小时。

Then, the tubes were charged with 16 zirconium ceramic beads (12 of 2.8 mm diameter and 4 of 6.5 mm diameter) and 25 mL mixture of methanol - ethyl acetate (1:2, v/v). Tubes were securely sealed with matching caps and the fungal mass was pulverized in an OMNI Bead Ruptor at 5.5 m/sec for 45 s. The slurry was centrifuged at 1,800 × .

然后，向管中加入16个锆陶瓷珠（12个直径为2.8毫米，4个直径为6.5毫米）和25毫升甲醇-乙酸乙酯混合物（1:2，v/v）。用匹配的盖子将管牢固密封，并将真菌块在OMNI珠破裂器中以5.5 m/sec的速度粉碎45 s。浆料以1800倍离心。

克

for 3 min in a Model 6755 LSE Compact Centrifuge (Corning Life Sciences); the supernatants were collected with glass pipettes into 250 mL round bottom flasks. Extraction/pulverization of the precipitates was repeated two more times with 20 mL ea. of the methanol - ethyl acetate mixture (1:2, v/v) followed by centrifugation and collection of the supernatants.

在6755型LSE紧凑型离心机（康宁生命科学）中放置3分钟；用玻璃移液管将上清液收集到250 mL圆底烧瓶中。沉淀物的提取/粉碎用20毫升ea重复两次。甲醇-乙酸乙酯混合物（1:2，v/v），然后离心并收集上清液。

The 3 extracts from the same samples were combined and evaporated with a Rotavapor-R rotary evaporator (Brinkmann Instruments, Westbury, NY) at 40 °C to dryness followed by addition of 10 mL of precisely measured methanol to each flask with dry residues. Then the flasks were capped with matching stoppers and sonicated in a Model T-9 ultrasonic bath (L&R, Kearny, NJ) for 30 s; the solution was transferred to 15 mL glass vials (Thermo Scientific, cat.

将来自相同样品的3种提取物合并，并用Rotavapor-R旋转蒸发器（Brinkmann Instruments，Westbury，NY）在40℃下蒸发至干燥，然后向每个烧瓶中加入10毫升精确测量的甲醇和干燥残留物。然后用匹配的塞子盖住烧瓶，并在T-9型超声波浴（L＆R，Kearny，NJ）中超声处理30秒；将溶液转移到15毫升玻璃小瓶（Thermo Scientific，cat。

# B7800-4) with matching caps and centrifuged in the same centrifuge under the same as above conditions for 3 min. About 0.25 mL aliquots of the centrifuged samples were filtered into 400 µL polypropylene autosampler vials (Fisher Scientific, cat. # C4010-11) through 10 × 10 mm pieces of glass fiber filter paper firmly placed into the narrow part of Pasteur pipettes; nitrogen gas from a compressed nitrogen tank was used to expedite filtration.

#B7800-4）带有匹配的盖子，并在与上述条件相同的离心机中离心3分钟。将约0.25 mL离心样品的等分试样通过10×10 mm玻璃纤维滤纸过滤到400µL聚丙烯自动进样器小瓶（Fisher Scientific，目录号C4010-11）中，将其牢固地放入巴斯德移液管的狭窄部分；来自压缩氮气罐的氮气用于加速过滤。

Vials were sealed with matching caps with PTFE septa (Fisher Scientific, cat. # C4010-60 A). Between 1/10th of a µL and 3 µL of filtered extracts were injected into the HPLC system..

小瓶用PTFE隔膜（Fisher Scientific，目录号#C4010-60 A）的配套盖密封。将1/10µL至3µL过滤后的提取物注入HPLC系统。。

HPLC-DAD-MS analysis

HPLC-DAD-MS分析

Separations of authentic standards and the fungal extracts were performed using a tandem HPLC − MS system equipped with Vanquish Split Sampler FT (part # VF-A10-A), Diode Array Detector FG (part # VF-D11-A) covering the 210–800 nm range, Quaternary Pump F (part # VF-P20-A) (all Thermo Scientific, San Jose, CA), and a 100 mm × 4.6 mm i.d., 3.5 μm XSelect HSS C18 analytical column (Waters).

使用配备有Vanquish Split Sampler FT（part#VF-A10-a），二极管阵列检测器FG（part#VF-D11-a）的串联HPLC-MS系统进行真实标准品和真菌提取物的分离，覆盖210-800 nm范围，四元泵F（part#VF-P20-a）（all Thermo Scientific，San Jose，CA）和100 mm×4.6 mm i.d.，3.5μmXSelect HSS C18分析柱（Waters）。

H。

O (A), MeOH (B), and 1% HCOOH in H

O（A）、MeOH（B）和1%HCOOH在H

O (C) were used in the following gradient: initial conditions, 31% A/65% B/4% C, changed linearly to 0% A/96% B/4% C in 4 min, held isocratic for 10 min, and then changed to initial conditions in 0.01 min and held for 3 min before the next injection. The flow rate was 0.5 mL/min. The column was maintained at 40 °C.

O（C）用于以下梯度：初始条件，31%A/65%B/4%C，在4分钟内线性变化至0%A/96%B/4%C，保持等度10分钟，然后在0.01分钟内变为初始条件，并在下次注射前保持3分钟。流速为0.5 mL/min。色谱柱保持在40℃。

MS analysis was performed using an LTQ XL ion trap mass spectrometer equipped with an APCI interface and operated with Xcalibur version 4.4.16.14 software (Thermo Scientific, San Jose, CA). The data were acquired in the full-scan mode (MS) from m/z 100 to 2000. The capillary temperature was 130 °C, APCI vaporizer temperature 230 °C, sheath gas flow 50 units, auxiliary gas flow 5 units, capillary voltage 9 V, and source voltage 6.0 kV.

使用配备有APCI接口并使用Xcalibur版本4.4.16.14软件（Thermo Scientific，San Jose，CA）操作的LTQ XL离子阱质谱仪进行MS分析。数据是在m/z 100至2000的全扫描模式（MS）下采集的。毛细管温度为130°C，APCI蒸发器温度为230°C，鞘气流量为50单位，辅助气体流量为5单位，毛细管电压为9 V，源电压为6.0 kV。

In MS.

在MS中。

analyses, the [M + H]

分析，[M+]

ions observed for each chromatographic peak in full-scan mode, were isolated and subjected to source collision induced dissociation (CID) using He gas. In all CID analysis, the isolation width, relative fragmentation energy, relative activation Q, and activation time were 1.0, 20 or 35%, 0.25, and 30 ms, respectively.

分离在全扫描模式下观察到的每个色谱峰的离子，并使用He气体进行源碰撞诱导解离（CID）。在所有CID分析中，隔离宽度，相对碎裂能，相对活化Q和活化时间分别为1.0、20或35%，0.25和30 ms。

Concentrations of all identified compounds in the extracts were calculated by reference to peak areas (calibration curves) of corresponding pure standards at their UV absorption maxima..

通过参考相应纯标准品在其紫外吸收最大值处的峰面积（校准曲线）计算提取物中所有鉴定化合物的浓度。。

Statistical analysis

统计分析

Results expressed in µg/g from five biological replicates for each compound identified were analyzed by analysis of variance (ANOVA), and means were compared by Tukey’s test using SYSTAT (Sigma Plot V. 15).

通过方差分析（ANOVA）分析鉴定的每种化合物的五个生物学重复的以µg/g表示的结果，并使用SYSTAT（Sigma Plot V。15）通过Tukey检验比较平均值。

Mating type

配合类型

Previously we reported that annotated Contig_298

以前我们报道过带注释的Contig\u 298

contains the MAT 1 locus

包含MAT 1轨迹

. Thus, we explored the possibility that color-CP isolates could have different mating types. For this, we first performed de novo assemblies of their genomes, and BLAST

因此，我们探索了彩色CP分离株可能具有不同交配类型的可能性。为此，我们首先对其基因组进行了从头组装，并进行了BLAST

of Contig_298 to those assemblies. Then, using Whole Genome Alignment (WGA)

这些组件的重叠群298。然后，使用全基因组比对（WGA）

with minimum initial seed 40 bp, and 100 bp minimum alignment block length, compared the BLAST results with the genomes of IPAVE0302

以最小初始种子40 bp，最小比对块长度100 bp，将BLAST结果与IPAVE0302基因组进行比较

and CBS 151044

和CBS 151044

. Further iterations of BLAST analysis of the MAT locus using

.使用MAT轨迹的BLAST分析的进一步迭代

Cladosporium fulvum

黄枝孢菌

Race 5 (Syn.

种族5（Syn。

Fulvia fulva

番茄叶霉病菌

) genome as a model

)基因组作为模型

in combination with WGA were performed to compare all six isolates and characterize the MAT1-1 and MAT1-2 MAT + sexual cell fertilization-promoting factors. All the analyses were performed using CLC Genomics Workbench 23.0.2.

结合WGA进行比较所有六种分离株并表征MAT1-1和MAT1-2 MAT++性细胞受精促进因子。所有分析均使用CLC Genomics Workbench 23.0.2进行。

Resistance to xenobiotics

对异生素的抗性

The effect of xenobiotics on growth area and density of CP isolates was evaluated. The four xenobiotics tested were: caffeine (CAF)(CAS Number: 58-08-2) Sigma-Aldrich, tebuconazole (TEB)(CAS Number: 107534-96-3), prothioconazole (PROT)(CAS Number: 178928-70-6), and chlorothalonil (CHLOR)(2,4,5,6-Tetrachloroisophthalonitrile; CAS Number: 1897-45-6), was tested at four different concentrations against the three CP color isolates and the reference NRRL 64,463.

评估了异生素对CP分离株生长面积和密度的影响。测试的四种异生素是：咖啡因（CAF）（CAS号：58-08-2）Sigma-Aldrich，戊唑醇（TEB）（CAS号：107534-96-3），丙硫唑醇（PROT）（CAS号：178928-70-6）和百菌清（CHLOR）（2,4,5,6-四氯间苯二甲腈；CAS号：1897-45-6），在四种不同浓度下针对三种CP颜色分离物和参考NRRL 64463进行测试。

Given its low solubility, CAF was added to hot PDA medium immediately after autoclaving and thoroughly mixed before pouring plates. The final concentrations used for CAF were 2, 4, 6 and 10 mM. TEB, PROT, and CHLOR were added after cooling to 55 °C the autoclaved PDA medium and thoroughly mixed before pouring plates.

鉴于其低溶解度，在高压灭菌后立即将CAF加入热PDA培养基中，并在浇注板之前充分混合。用于CAF的最终浓度为2、4、6和10 mM。将高压灭菌的PDA培养基冷却至55°C后加入TEB，PROT和CHLOR，并在浇注板之前充分混合。

The final concentrations used for TEB, PROT and CHLOR were 0.3, 1.0, 3.3 and 10.0 mg a.i./L. The CP isolates grown on PDA medium were used as controls. For testing the response to xenobiotics, the CP isolates were placed on top of Polyvinylidene fluoride (PVDF) membranes 0.45 μm (HVLP04700, Merck Millipore, Cork, Ireland).

用于TEB，PROT和CHLOR的最终浓度分别为0.3、1.0、3.3和10.0 mg a.i./L。在PDA培养基上生长的CP分离株用作对照。为了测试对异生素的反应，将CP分离株置于0.45μm聚偏二氟乙烯（PVDF）膜（HVLP04700，默克密理博，科克，爱尔兰）的顶部。

The PVDF membranes were autoclaved for 10 min before use, then single membranes were placed on the surface of the medium of each plate. One cm.

使用前将PVDF膜高压灭菌10分钟，然后将单个膜放置在每个板的介质表面上。一厘米。

of 12-day old culture grown on PDA was placed in sterile test tubes containing 2 mL sterile distilled water then ground with a LabGEN 125 Tissue Homogenizer. For each concentration of xenobiotic and fungal isolate, three plates were prepared placing four undisturbed 20-µL-drops of homogenate on each PVDF membrane.

将在PDA上生长的12天大的培养物置于含有2 mL无菌蒸馏水的无菌试管中，然后用LabGEN 125组织匀浆器研磨。对于每种浓度的异生素和真菌分离物，制备三个平板，在每个PVDF膜上放置四滴未受干扰的20µL匀浆。

The plates were incubated in darkness at 26 °C for 5 days, then three randomly selected drops were photographed using backlight in a stereoscope Nikon SMZ800 and Leica Application Suite software (LAS V4.3). Areas and density of growth for each drop were estimated using ImageJ software (.

将板在26℃的黑暗中孵育5天，然后在立体镜Nikon SMZ800和Leica Application Suite软件（LAS V4.3）中使用背光拍摄三滴随机选择的液滴。使用ImageJ软件估算每滴的面积和生长密度（。

https://imagej.net/ij/

, National Institutes of Health, USA) as described before

，美国国立卫生研究院），如前所述

. The experiments were repeated once, data were analyzed by ANOVA using SYSTAT V.15 and means compared by Tukey’s test.

重复实验一次，使用SYSTAT V.15通过ANOVA分析数据，并通过Tukey检验比较均值。

Data availability

数据可用性

The datasets generated and/or analyzed during the current study are available in NCBI GenBank, genomes SRA: SRR24707293, SRR24718870, SRR24718913; transcriptomes SRA: SRR25052186, SRR25057223, SRR25054762, SRR25144991, SRR25182278, SRR25231499, SRR25234020, SRR25237168. Further details listed in Supplementary Tables 1 and 3..

在当前研究期间生成和/或分析的数据集可在NCBI GenBank中获得，基因组SRA:SRR24707293，SRR24718870，SRR24718913；转录组SRA:SRR25052186，SRR25057223，SRR25054762，SRR25144991，SRR25182278，SRR25231499，SRR25234020，SRR25237168。补充表1和3中列出了更多详细信息。。

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Acknowledgements

致谢

All those who contributed to the manuscript are in the list of authors.

所有为手稿做出贡献的人都在作者名单中。

Author information

作者信息

Author notes

作者笔记

Renee S. Arias and Victor S. Sobolev contributed equally to this work.

Renee S.Arias和Victor S.Sobolev对这项工作做出了同样的贡献。

Authors and Affiliations

作者和隶属关系

USDA-ARS National Peanut Research Laboratory, 1011 Forrester Dr. S.E, 39842, Dawson, GA, USA

美国农业部花生研究所国家花生研究实验室，1011 Forrester Dr.S.E，39842，道森，佐治亚州，美国

Renee S. Arias, Valerie A. Orner, Travis E. Walk, Alicia N. Massa, Pirada S. Higbee, Marshall C. Lamb & Victor S. Sobolev

蕾妮·S·阿里亚斯、瓦莱丽·A·奥尔纳、特拉维斯·E·沃克、艾丽西亚·N·马萨、皮拉达·S·希比、马歇尔·C·兰姆和维克多·S·索博列夫

Valdosta State University, 1500 N. Patterson St, Valdosta, GA, 31698, USA

瓦尔多斯塔州立大学，1500 N.Patterson St，瓦尔多斯塔，佐治亚州，31698，美国

Emily G. Cantonwine & Atalya Manchester

Emily G.Cantonwine和Atalya Manchester

Department of Agricultural Biology, Colorado State University, 301 University Ave, Fort Collins, CO, USA

科罗拉多州立大学农业生物学系，美国科罗拉多州柯林斯堡大学大道301号

Jane E. Stewart & John T. Dobbs

简·E·斯图尔特和约翰·T·多布斯

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Renee S. Arias

蕾妮·S·阿里亚斯

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艾米丽·G·坎顿温

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瓦莱丽A.文件夹

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Travis E. Walk

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Alicia N. Massa

艾丽西娅·N·马萨

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Jane E. Stewart

简·E·斯图尔特

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John T. Dobbs

约翰·T·多布斯

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Atalya Manchester

阿塔利亚曼彻斯特

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Pirada S. Higbee

皮拉达S.希比

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Marshall C. Lamb

马歇尔C.兰姆

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Victor S. Sobolev

维克多·S·索博列夫

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Contributions

捐款

Author contributions: EGC, RSA, VSS: conceptualization, experimental design, data analysis, original draft preparation; JES, JTD, ANM: genome annotation, parsing, important suggestions; VSS, TEW, PSH: chemical profiling and formal analysis; VAO, TEW, VSS: data acquisition, uploading sequences to NCBI, long term storage of cultures; EGC, AM: isolation, morphological characterization, microscopy; MCL, RSA, VSS: funding acquisition; all authors reviewed, discussed and approved the final version of the manuscript..

作者贡献：EGC，RSA，VSS：概念化，实验设计，数据分析，原稿准备；JES，JTD，ANM：基因组注释，解析，重要建议；VSS，TEW，PSH：化学分析和形式分析；VAO，TEW，VSS：数据采集，将序列上传到NCBI，长期保存文化；EGC，AM：分离，形态表征，显微镜；MCL，RSA，VSS：资金收购；所有作者都审查，讨论并批准了稿件的最终版本。。

Corresponding author

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Renee S. Arias

蕾妮·S·阿里亚斯

Ethics declarations

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Competing interests

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The authors declare no competing interests.

作者声明没有利益冲突。

Ethics declarations

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This work does not include the use of animals or humans.

这项工作不包括使用动物或人类。

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Electronic supplementary material

电子补充材料

Below is the link to the electronic supplementary material.

以下是电子补充材料的链接。

Supplementary Material 1

补充材料1

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Arias, R.S., Cantonwine, E.G., Orner, V.A.

Arias，R.S.，Cantonwine，E.G.，Orner，V.A。

et al.