Fungi-metabolites production and networking analysis in saprophytes and pathogens fungi

doi:10.21203/rs.3.rs-5248703/v1

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Fungi-metabolites production and networking analysis in saprophytes and pathogens fungi

https://doi.org/10.21203/rs.3.rs-5248703/v1

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Fungi are important parasitic microorganism of plants, which mainly include saprophytes and pathogens species. However, differences of mycotoxins production by different fungi still remain challenging. To address this bottleneck, saprophytes and pathogens Alternaria spp. are used for inoculation and metabolites production analysis are process via liquid chromatography- high resolution mass spectrometry (LC-HRMS). Characteristic neutral loss is present for modified mycotoxins screening and feature fragments which match chemical similarity are applied for annotation. The results revealed that Saprophytic Alternaria spp. are the dominant secondar metabolites producing fungi with extremely differences. Glucose conjugations, ALU-G, AME-G, and TeA-G, are identified as modified mycotoxins. In addition, molecular networking result reveals that metabolic pathway of Alternaria mycotoxins mainly include acetylation, dehydration, didehydration, dimethylamination, hydrogenation, isomerization, methylation, combination of methylation and hydroxylation, and combination of demethylation and dihydroxylation.

Biological sciences/Biotechnology

Biological sciences/Microbiology

Alternaria species are known as major plant saprophytes and pathogens and belongs to a diverse and ubiquitous group of fungi. Pathogen Alternaria are reported to induce black spot disease in plants during the farming and postharvest storage and preservation(1). Black spot disease could bring severe yield reductions in crops and fruits, such as cabbage, broccoli, pears, et al(2, 3), which might bring huge economic loss. The fungus of saprophytic Alternaria spp. is reported with strong parasitic and saprophytic abilities in order to obtain energy by decomposing cellulose(4, 5). Saprophytic Alternaria spp. fungi are commonly involved in fungal spoilage, which might also bring severe toxic risks to Agriculture and food industry.

Fungi are well-known as producers of diverse secondary metabolites, including hazardous mycotoxins products. These potential mycotoxins are toxic to invertebrates and vertebrates including human beings. For a long time, mycotoxin productions have been reported during storage conditions due to saprophytic mold growth on grains(6, 7). Recently, it is also reported that mycotoxins could be produced by pathogenic fungi during the development of diseases on plants(8, 9).

After infection by Alternaria species, some low-molecular-weight secondary metabolites including different Alternaria mycotoxins could be produced and contaminated in fruits. Till now, five class of Alternaria mycotoxins have been detected and identified, including dibenzo-α-pyrones (represented by alternariol (AOH), and alternariol monomethyl ether (AME)), perylene quinones (represented by altertoxins I-III (ATX I-III)), tetramic acid derivatives (represented by tenuazonic acid (TeA)), miscellaneous structures (represented by tentoxin, (TEN)), and aminopentol esters (represented by A. alternata f. sp. lycopersici toxins (AAL toxins)).

Beside these free mycotoxins, conjugated Alternaria mycotoxins have also been detected and identified(10). Glucose and sulfated conjugations of AME, AOH, TeA, et al. are confirmed as major modification form of Alternaria mycotoxins(11). And these conjugated mycotoxins are also detected naturally contaminated in foods and feeds(12, 13). These conjugated mycotoxins will be transformed to free mycotoxins in digestive tract and might bring severe hazardous effects to consumers(14, 15).

Pear is an important temperate-climate fruit, and it is cultivated since the beginning of civilization due to the delicacy of its flavor(16). Nowadays, pear has become an important commodity in Europe, United States, and China. Pear fruits are high susceptible to numerous fungi due to the high-water activity, rich of nutrients, and thin skins, especially for Alternaria spp.(17). Alternaria spp. are regarded as major plant related fungi. These Alternaria spp. fungi are usually divided into two categories in pears, saprophytes and pathogen fungi(18). Alternaria mycotoxins contaminated in pear fruit and related foodstuff have been reported in Alternaria spp. infected samples, including ATX-I, ALT, AME, AOH, TEN, TeA, and other mycotoxins(19).

Certain species in the genus Alternaria produce host-specific toxins (HSTs) that might be related to their pathogenicity and virulence(20). HSTs play a role in determining the host range of specificity of plant pathogens at low very concentrations(21). Previous research demonstrated the level of pathogenicity of different isolates recovered from different plant samples and their efficiency for mycotoxin production. The difference and variation of mycotoxin production might be related to defense mechanism responded by plants or to some extent their resistance against the Alternaria pathogen and disease.(22, 23)

Regarding pear fruits, as far as we aware, there are no close studies focused on differences of mycotoxins production in fungi incidence between pathogenic and saprophytic Alternaria spp. This work aims to explore critical secondary metabolites in Alternaria fungi inoculated pear fruit samples. Specially, the incidence of mycotoxins production between pathogenic and saprophytic Alternaria spp. are also investigated and compared. This work will be benefit for better understanding of the role of mycotoxins plays in pathogenicity of Alternaria spp. during the process of plant infection.

Morphological results in saprophyte and pathogen Alternaria spp. inoculated pear samples.

Pear samples were inoculated with saprophyte and pathogen Alternaria spp. and stored at 4℃ (cold storage) and 25℃ (room temperature). Morphological results are shown in Fig. 1. From the figure, spots on inoculated pear samples with both saprophyte and pathogen strains gradually increase in size and the decay of pears get worse following the storage time after inoculation at 25 ℃. Obvious black spot could be observed at 3rd day after inoculation, and pear fruits could be sampled for only four times (20 days) due to the severe decay. As to the storage at 4 ℃, black spots slightly increase after inoculation, and pear fruits could be stored for 30 days with 6 sampling times. Morphological results of pears in different storage temperature show that cold storage condition could inhibit both saprophyte and pathogen Alternaria spp. while 25℃ is not suitable for the storage of infected pears.

From the morphological results, both saprophyte and pathogen Alternaria spp. could infest pear fruits and cause black rot to pears during the storage at 25℃. However, morphological results show no obvious differences between saprophyte and pathogen Alternaria spp. inoculated pear samples both at 4℃ (cold storage) and 25℃ (room temperature).

Natural Alternaria mycotoxins Production Investigation

Although morphological results show unobvious differences between saprophyte and pathogen Alternaria spp. inoculated pear samples. However, natural Alternaria mycotoxins productions still arouse the interest of researchers. In this work, natural Alternaria mycotoxins products were screened and identified with high resolution mass spectrometry (HRMS).

Among all potential natural Alternaria products, the seven target mycotoxins with commercial standards (ALT, ALU, AME, AOH, ATX-I, TeA, and TEN) qualitative and quantitative analyzed. According to ionization mode and molecular weight, one protonated precursor of each target [M + H]⁺ is selected. Based on MSMS spectrum of precursor, three preponderant product ions were selected, which meet the requirement for the Regulation (EU) 2021/808(24). Chromatograms and mass spectrum parameters for the seven Alternaria mycotoxins are shown in Fig. 2. In this work, satisfactory peak shape and response are obtained with no obvious interfere peaks at the retention time. And the seven Alternaria mycotoxins in the two sample groups and one control group at 4°C and 25°C were quantitatively analyzed based on the peak area (results shown in Table 1).

Table 1

Alternaria mycotoxins production in *saprophyte spp.* (Group P) and *pathogen spp.* (Group S) inoculated pear samples during the storage at 4℃ and 25℃.
Group	Strain	Treatment	ALT (µg/kg)	ALU (µg/kg)	AME (µg/kg)	AOH (µg/kg)	ATX-I (µg/kg)	TeA (µg/kg)	TEN (µg/kg)
P	HB-11	4℃ 0 ~ 30 d	ND	ND	53 ~ 307	11 ~ 76	18 ~ 1,005	8 ~ 5466	ND
	HB-27		ND	ND	9 ~ 11	ND	27 ~ 1,409	8 ~ 7532	ND
	HB-72		ND	ND	8 ~ 15	ND	ND	65 ~ 2398	ND
	HB-122		ND	ND	52 ~ 198	9 ~ 44	ND	12 ~ 1908	2 ~ 13
	HB-196		ND	ND	47 ~ 215	27 ~ 149	14 ~ 560	22 ~ 1349	ND
S	M1-2		24 ~ 181	48 ~ 265	150 ~ 15,607	43 ~ 1,034	17 ~ 150	209 ~ 41540	113 ~ 1309
S	B2-1-2		32 ~ 251	29 ~ 297	399 ~ 10,718	29 ~ 500	ND	1047 ~ 62263	75 ~ 2252
P	HB-11	25℃ 0 ~ 20 d	63 ~ 330	14 ~ 122	324 ~ 2,620	25 ~ 575	971 ~ 1,536	43 ~ 198	ND
	HB-27		ND	ND	362 ~ 2,902	7 ~ 347	692 ~ 2,019	63 ~ 276	ND
	HB-72		ND	47 ~ 496	599 ~ 4,707	8 ~ 266	1251 ~ 1,701	64 ~ 292	ND
	HB-122		18 ~ 57	12 ~ 479	240 ~ 3,472	88 ~ 445	1099 ~ 1,326	62 ~ 118	22 ~ 49
	HB-196		78 ~ 106	21 ~ 316	418 ~ 5,328	13 ~ 207	744 ~ 2,596	40 ~ 231	21 ~ 76
S	M1-2		457 ~ 7,904	237 ~ 4,238	396 ~ 13,540	129 ~ 2,416	13 ~ 196	822 ~ 358085	53 ~ 577
S	B2-1-2		910 ~ 9,876	248 ~ 5,157	497 ~ 19,381	130 ~ 3,388	ND	2038 ~ 204752	121 ~ 880
ND: not detected. S: saprophyte. P: pathogen.

During the storage at 25°C, AOH, AME, and TeA are detected in all saprophyte and pathogen Alternaria spp. inoculated pear samples. The highest concentrations in saprophytes inoculated pear samples (group S) are 9,876, 5,157, 19,381, 3,388, 204,751, and 880 µg/kg for ALT, ALU, AME, AOH, TeA, and TEN in strain B2-1-2, while the highest concentrations are 7,904, 4,238, 13,540, 2,416, 96, 358,085, and 577 µg/kg for ALT, ALU, AME, AOH, ATX-I, TeA, and TEN in strain M1-2. Specially, ATX-I is detected in strain M1-2 with concentration range at 13 ~ 196 µg/kg, while it is not detected in strain B2-1-2 in group S. Concentrations of ATX-I in group P are detected with highest concentration at 2,596 µg/kg, which is 10 times of that group S. Concentration of ALT in group P are less than 5% of that in group S, and it is even not detected in strain 27 and 72. ALU is almost 10% of group S, and it is still not detected in strain 27. Concentrations of AME and AOH are almost 30% and 15% of group S. Trace amount of TeA are detected at one thousandth of group S. As to TEN, it is only detected in strain 122 and 196 with concentrations at 10% of group S.

Here comes to the storage at 4°C, ATX-I is different from other six mycotoxins with highs concentrations detected in Group P as storage at 25°C. AME and TeA are detected in all saprophyte and pathogen Alternaria spp. inoculated pear samples. The highest concentrations are detected in Group S at 15,607 and 62,263 µg/kg, while concentrations of the two are almost only 2% of Group P. Trace amount of AOH and TEN are detected only in some of pathogen strains (3 strains for AOH and 1 strain for TEN in Group P). It is also observed that ALT and ALU are free in all pathogens inoculated pear samples.

Above all, apparent differences of Alternaria mycotoxins are observed between saprophyte and pathogen Alternaria spp. inoculated pear samples. Compared to pathogens, saprophyte strains are preponderant Alternaria mycotoxins production fungi in inoculated pear samples, except for ATX-I. In addition, the results also show that concentrations of target Alternaria mycotoxins are higher during the storage at 25°C than 4°C.

Modified metabolites of Alternaria mycotoxins Investigation

In Full Mass and ddMS2 scan of this research, ddMS2 scan of TOP 5 precursors is acquired for natural Alternaria products analysis. MS1 and MS2 spectrum in HRMS are processed for neutral loss screening with MZmine (http://mzmine.github.io/). According to the preliminary test, glucose conjugated products are observed in this research. Therefore, glucose conjugates as well as the unconjugated natural products are investigated in this work.

The neutral loss of glucose conjugates in HRMS is 162.0528 Da according to the chemical formula, which losing one anhydro glucose group in chemical structure. NLF (neutral loss filtering) function is used for glucose conjugated metabolites screening in this work by setting neutral loss at 162.0528 Da (Fig. 3A and table S1).

The screening results show that totally 72 glucose conjugates are obtained. And glucose binding reaction is preponderate in pathogen (60 glucose conjugates) than saprophyte (32 glucose conjugates). Among them, there are 20 common glucose conjugates observed in both pathogen and saprophyte. The screening results are further identified with MS2 spectrum based on accuracy mass spectrum using in-house Vender Xcalibur software. In this work, ALU, AME, and TeA glucose conjugates are identified in Alternaria spp. inoculated pear samples (Fig. 3B). Trace amount of ALU-G and AME-G are only detected in both Group S and Group P during the first 15 days of storage, while large amount of TeA-G is detected in Group S only.

Based on the previous seven natural Alternaria mycotoxins and three glucose-conjugated Alternaria metabolites production investigation results, heatmap was established for the screening results (Fig. 3C). From the figure, high amounts of these hazardous products are detected at 25℃. However, cold storage at 4℃ could not guarantee safety due to that Alternaria mycotoxins could also be detected. High amounts of most Alternaria mycotoxins are observed in saprophyte infected pears, except for AME-G, ALU-G, and ATX-1. TeA-G exhibit the highest response in HRMS in saprophyte only.

Natural products differences between Pathogenic and Saprophytic Alternaria spp. inoculated pears

In this work, natural products differences between Pathogenic and Saprophytic Alternaria spp. inoculated pear samples are investigated with HRMS. Features of natural products were processed with mass detection function in MZmine for Full mass and ddms2 filtering. And chromatograms of filtered features were re-established with chromatogram builder function. These chromatograms were further filtered and aligned with function of deconvolution, isotope peak group, and join aligner. Finally, files containing full mass and ddms2 data of all filtered features were exported for further analysis.

Volcano plots between Pathogenic (Group P) and Saprophytic (Group S) Alternaria spp. inoculated pears are constructed via fold change (FC) values (Pathogenic/Saprophytic) and p values based on peak areas of processed features in previously exported full mass data file. In this study, natural products differences with fold change ≥ 4.0 (|log₂(FC)| ≥ 2.0) and p value ≤ 0.05 and 0.01 (T test) are considered statistically significant and very significant different, respectively. The vertical lines represent 4.0-fold up- and down-regulation between Pathogenic and Saprophytic Alternaria spp., and the horizontal lines are related to p-value at 0.01 and 0.05 (25).

As shown in Fig. 4, totally 36 natural products exhibit differences between Pathogenic and Saprophytic Alternaria spp. (|log₂(FC)| ≥ 2.0, P < 0.05). From the figure, Saprophytic Alternaria spp. are the dominant products producing fungi which could produce more products with extremely differences. Totally, 35 natural products including seven Alternaria mycotoxins are detected up-regulated in the Saprophytic (Group S), while only one feature is detected up-regulated in the Pathogenic (Group P) (log₂(FC) ≤ -2.0, P < 0.05). Specially, seven Alternaria mycotoxins are detected upregulated in saprophytic Alternaria spp. (Group S), including TEN (Log₂(FC) = -6.00, P = 2.06E-07), TeA-G (Log₂(FC) = -10.08, P = 1.82E-04), AOH (Log₂(FC) = -2.13, P = 3.22E-03), AME (Log₂(FC) = -2.00, P = 5.00E-03), ALT (Log₂(FC) = -2.47, P = 8.71E-03), TeA (Log₂(FC) = -4.62, P = 2.51E-02), ALU (Log₂(FC) = -2.17, P = 4.66E-02). As to the other three Alternaria mycotoxins, ALU-G, AME-G, and ATX-I, the Differences between Pathogenic and Saprophytic are not obvious with Log₂(FC) less than 2 times fold up and P value > 0.1.

Molecular networking based metabolic pathway analysis for Alternaria mycotoxins

Feature based molecular networking (FBMN) in GNPS platform (https://gnps.ucsd.edu/) is applied for visible metabolic pathway analysis of Alternaria mycotoxins production in Pathogenic (Group P) and Saprophytic (Group S) Alternaria spp. inoculated pears. Files containing full mass and ddms2 data processed in section 2.3 are uploaded and analyzed with FBMN. Parameters are settled with a minimum requirement of 6 ions and cosine score over 0.7. Metadata file containing Pathogenic or Saprophytic fungi information was added to show the experimental setup which could be benefit for better downstream data visualization, analysis and interpretation(26, 27).

The natural production results of Alternaria mycotoxins are generated and exported to Cytoscape software for visualized analysis. In molecular networking results, Alternaria mycotoxins and potential metabolites are connected to each other, which refer to "molecular families". The connected edge represents as mass shift, which indicating metabolic pathway of these products(28–30) (Fig. 5). Each node represents a compound, which is divided by the two different Alternaria fungi with different colors to distinguish the spectral sources.

From the figure, six Alternaria mycotoxin families (AOH and AME; ALT; ALU; TEN; TeA-G; and TeA) are obtained with metabolic pathway and three (ATX-I, ALU-G, and AME-G) are detected with one node only. Based on the pie results in the figure, it can be observed that Saprophytic (Group S) Alternaria spp. are predominant for mycotoxins and potential metabolites production.

Family A consists of four compound nodes, including AOH (C₁₄H₁₀O₅, m/z: 259.0601, RT: 5.84) and AME (C₁₅H₁₂O₅, m/z: 273.0757, RT: 6.94) and two metabolites m/z: 273.0756 (RT: 7.03 min) and m/z: 289.0705 (RT: 6.29 min) (Fig. 5A). AOH and AME are connected to each other with mass shift at 14.016, indicating AME is methylation metabolite of AOH with high reliability (cosine value over 0.75). Feature with m/z: 273.0756 is potential metabolite of AOH with mass shift at 14.016, indicating metabolic pathway of methylation. And it is also an isomer of AME. Feature with m/z: 289.0705 is identified as metabolite of AOH. And the metabolic pathway is combination of methylation and hydroxylation. This metabolite is also identified in the GNPS library as 2-Hydroxyalternariol-9-methyl ether_120110 (C₁₅H₁₂O₆).

Three features make up family B (Fig. 5B), including ALT (C₁₅H₁₆O₆, m/z: 293.1518, RT: 5.22). The three features directly connect with each other. The feature node (C₁₅H₁₄O₅, m/z: 275.0913, RT: 5.06 min) is a dehydration metabolite of ALT with mass shift at 18.060. Further hydrolysis reaction result in the feature node (C₁₅H₁₆O₆, m/z: 293.1016, RT: 4.88), which is also an isomer of ALT.

Family C of ALU (C₁₅H₁₆O₆, m/z: 291.0867, RT: 5.60) is shown in Fig. 5C, which consists of four features. Two isomers ((m/z: 291.0886, RT: 5.71) and (m/z: 291.0882, RT: 4.83)) and one dehydration metabolite (m/z: 273.0757, RT: 5.71) of ALU are detected in the molecular networking result.

Family D consists of five feature nodes (Fig. 5D). The TEN node (C₂₂H₃₀N₄O₄, m/z: 415.2323, RT: 5.97) is directly connected to the other four compound nodes. Metabolite (m/z: 460.2908, RT: 5.52) of TEN exhibits a mass shift of 45.059 with delta mmu of 1.151, indicating dimethylamination reaction. Further dedimethylamination reaction results in a TEN isomer (m/z: 415.2535, RT: 5.67). Mass shift at 2.017 indicates the metabolic pathway of hydrogenation. Metabolite (m/z: 327.1961, RT: 5.79) with mass shift at 88.036 is not identified in this work.

Family E and F are TeA-G (C₁₀H₁₅NO₃C₆H₁₀O₅, m/z: 360.1653, RT: 4.61) and TeA cluster (C₁₀H₁₅NO₃, m/z: 198.1125, RT: 5.91), respectively (Fig. 5E, 5F). TeA-G family consists of 8 nodes. Six metabolites are detected with metabolic pathway mainly include methylation, dehydration, didehydration, and combination of demethylation and dihydroxylation with mass shift at 14.015, 18.010, 36.022, and 30.013, respectively. One feature (m/z: 210.1166, RT: 4.56) with mass shift at 84.014 is not identified in this work. TeA family consists of 5 nodes with two identified and two unidentified metabolites of TeA. Metabolic pathway of the two identified metabolic are isomerization and acetylation. Specially, acetylation reaction is only detected in the metabolism of TeA.

As shown in Fig. 5G, ATX-I (C₂₀H₁₆O₆, m/z: 353.1108, RT: 5.48) and two glucose-conjugated Alternaria mycotoxins ALU-G (C₂₀H₁₆O₆C₆H₁₀O₅, m/z: 453.1380, RT: 4.18) and AME-G (C₁₅H₁₂O₅C₆H₁₀O₅, m/z: 435.1596, RT: 4.61) are independent compound nodes without molecular networking. The results revealed that the three mycotoxins are difficult to be metabolized in inoculated pear samples.

According to the previous, Alternaria mycotoxins and related metabolites are identified based on molecular networking results. And metabolic pathways mainly include acetylation, dehydration, didehydration, dimethylamination, hydrogenation, isomerization, methylation, combination of methylation and hydroxylation, and combination of demethylation and dihydroxylation. Moreover, from the pie results in the molecular networking, it can be observed that Saprophytic Alternaria spp. are predominant for mycotoxins and potential metabolites production.

Pear has become an important commodity in most part of the world, including the Europe, United States, and China. However, due to the high-water activity, rich of nutrients, and thin skins, pear fruits are susceptible to fungal infections. Fungal infection might also result in the contamination of potential secondary metabolites in pears. Among all potential infection fungi, Alternaria spp. are known as major plant saprophytes and pathogens fungi. Pathogen Alternaria spp. could induce severe black spot disease in infected pears(31). Saprophytic Alternaria spp. is reported with strong parasitic and saprophytic characters(32)^,(33).

Most plant-related pathogen and saprophytic could produce mycotoxins after infection, which might be toxic to humans. The production of mycotoxins may present a fitness gain for the fungi. However, relationship between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors(34). Alternaria spp. is a plant related fungi that could synthesizes mycotoxins after infection in pears and other plants. Mycotoxins play roles in pathogenesis have been illustrated plants sensitive to the mycotoxins are also susceptible to the fungi(35).

The hazardous mycotoxins produced by plant-associated fungi have also been implicated in pathogenicity. Specially, fungi responsible for mycotoxin production are usually endophytes and saprophytes, which could infect and colonize living plant tissues. Accumulation of mycotoxins may be related with development of plant disease symptoms. Mycotoxins may be also detected even in the subtle biological effects of disease symptoms(36). In the morphological results of this research, both saprophyte and pathogen Alternaria spp. could infest pear with same biological effects with no obvious morphological differences.

During the storage at 25°C, Concentrations of ALT, ALU, AME, AOH, TeA, and TEN are only detected saprophyte strains in at 5%, 10%, 30%, 15%, 1‰, and 10% than that in pathogen strains, respectively. As to the storage at 4°C, saprophyte strains are also the dominant fungi for mycotoxins production with ALT and ALU are only detected in Saprophyte strains inoculated pear samples. Alternaria mycotoxins production between saprophyte and pathogen Alternaria spp. are quite different. Saprophyte strains are preponderant for most mycotoxins production fungi in inoculated pear samples.

In previous, sulfated and glucose conjugated modified Alternaria mycotoxins have been identified and reported (37, 38). Sulfated metabolites were reported in culture plate and fruits(39, 40), while glucose conjugated metabolites were mainly in infected foods and related products (41, 42). In this work, three glucose-conjugated mycotoxins metabolites were identified in infected pear samples, including TeA-G, ALU-G, and AME-G. Our work keeps correspondence with previous literature in Alternaria spp. infected apple and cherry samples (12, 25, 43). Among the three glucose-conjugated metabolite, TeA-G is up-regulated in the Group S with significant differences (P < 0.05) while ALU-G, AME-G, and ATX-I are detected without significant differences between Group S and Group P (P > 0.05).

Chemicals, solvents and reagents

Altenuene (ALT), Altenusin (ALU), Alternariol Monomethyl Ether (AME), Alternariol (AOH), Altertoxin-I (ATX-I), Tenuazonic Acid (TeA), and Tentoxin (TEN) standards (purity ≥ 98%) were purchased from Pribolab Pte Ltd. (Immunos, Singapore) (Chemical structures are shown in Figure S1).

Potato dextrose agar (PDA) culture mediums were purchase from Beijing Land Bridge Technology Co., Ltd. (Beijing, China). LC-MS grade methanol and acetonitrile were purchased from Dima Technology Inc. (Muskegon, MI, USA). HPLC grade Formic acid was purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA).

Apparatus

An Ultimate 3000 pumping system coupled to a Thermo Q/Exactive plus in positive electrospray mode (ESI⁺) was used in this work. Gradient elution program with run time of 12 min was used for chromatographic separation natural products as shown in table S2. 0.05% ammonia water containing 0.1% formic acid was performed as eluent A, while acetonitrile containing 0.1% formic acid as eluent B. A data-dependent acquisition (DDA) mode in HRMS was operated for analysis of each sample.

Full mass scan parameters were as follows: Spray voltage 3.8 KV; sheath gas pressure 40.00 arbitrary (Arb); S-lens RF level 50.00 and capillary temperature 320.00°C. The most optimized full mass parameters of DDA mode were as follows: scan range 120–900 m/z; Resolution 70,000; max injection time (IT) 100 ms; automatic gain control (AGC) target 3e⁶.

ddMS2 parameters of DDA mode was as follows: TOP N = 5, resolution 17,500; loop count 10; automatic gain control (AGC) target 1e5; maximum IT 50 ms; isolation window 1.5 m/z, normalized collisional energy (NCE) at 20, 40, and 70 eV; intensity threshold, 1.6e⁵; apex trigger, 3–8 s; dynamic exclusion, 6.0 s.

Fungi strains

Pathogenic Alternaria spp. strains (HB-1, HB-11, HB-27, HB-72, HB-122, and HB-196) were kindly supplied from Huazhong Agricultural University, while saprophytic Alternaria spp. strains (B2-1-2 and M1-2) were isolated and identified in our lab in Yantai University.

In vivo Test

Pathogenic and saprophytic Alternaria spp. strains were cultured in PDA medium for 7 days in alternated light and dark at 28°C before inoculation to pear samples. Spores were harvested by washing the culture in Petri dishes with 5 mL of sterile water after 7-d culture. Then, unnecessary mycelia were filtered out and spore suspension was adjusted to 1.5×10⁶ spores/mL.

Fresh pear samples were divided by two sample groups and one control group. The two sample groups were inoculated at the equator of the fruit with prepared pathogenic (Group P) and saprophytic (Group S) Alternaria spp. (spore concentration at about 10⁶/L), respectively. Then, pears were placed at 4°C and 25°C, and pears were sampled at every five days in 30 days totally for Alternaria mycotoxin analysis with each in triplicate. Samples in control group (Group C) were inoculated with sterile saline solution and sampled at the same condition as sample groups.

Sample Preparation for secondary metabolites Analysis

Pears were homogenized for potential mycotoxins and related metabolites extraction after sampling. 2 ± 0.02 g of homogenized samples were transferred to a 50 mL centrifuge tube. Then, 4 mL of ethyl acetate was added and vortexed for 30 s, and then shake for 30 min under 35°C for intermediate polar and non-polar products extraction. After centrifugation at 10,000 g for 5 min at 4°C, the supernatant liquid sample was transferred to a new 10 mL centrifuge tube. Potential high-polar products in the residue were re-extracted with 4 mL of Acetonitrile and the supernatant was combined with the previous after centrifugation at the same condition. The combined supernatants were taken to dryness under gentle nitrogen at 60°C and the residue was reconstituted with 1 mL of 75% methanol. Finally, each sample was filtered through 0.22 µm micropore cellulose membrane filter before HRMS analysis.

COMPETING INTERESTS

The authors declare no competing interests.

SUPPLEMENTARY INFORMATION

Supplementary information is available online.

Funding

for this project was provided by the Natural Science Foundation of Shandong Province (ZR2023MC097; ZR2022MC214) and National Natural Science Foundation of China (No. 31871718)

Author Contribution

X.M., L.W., and Y.L. conceived and designed all experiments. X.L. and S.L. performed the experiments. X.M., X.L. and Y.Y. analyzed the data. X.L. and Y.L. wrote the manuscript. S.Z. contributed reagents and administrative assistance. Y.W. and Y.L provided funding, and supervision.

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No competing interests reported.

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Fungi-metabolites production and networking analysis in saprophytes and pathogens fungi

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