A new brown rot disease of plum caused by Mucor xinjiangensis sp. nov. and screening of its chemical control

A novel species of Mucor was identified as the causal agent of a brown rot of Prunus domestica (European plum), widely grown in the south of Xinjiang, China. This disease first appears as red spots after the onset of the fruits. With favorable environmental conditions, fruit with infected spots turn brown, sag, expand, wrinkle, and harden, resulting in fruit falling. Fungal species were isolated from infected fruits. A phylogenetic analysis based on internal transcribed spacer (ITS) regions and the large subunit (LSU) of the nuclear ribosomal RNA (rRNA) gene regions strongly supported that these isolates made a distinct evolutionary lineage in Mucor (Mucoromycetes, Mucoraceae) that represents a new taxonomic species, herein named as Mucor xinjiangensis. Microscopic characters confirmed that these strains were morphologically distinct from known Mucor species. The pathogenicity of M. xinjiangensis was confirmed by attaching an agar disk containing mycelium on fruits and re-isolation of the pathogen from symptomatic tissues. Later, fourteen fungicides were selected to determine the inhibitory effect on the pathogen. Further, results showed that difenoconazole had the best effect on the pathogen and the strongest toxicity with the smallest half maximal effective concentration (EC50) value, followed by a compound fungicide composed of difenoconazole with azoxystrobin, mancozeb, prochloraz with iprodione, pyraclostrobin with tebuconazole, and trifloxystrobin with tebuconazole and ethhylicin. Present study provides the basis for the prevention and control of the novel plum disease and its pathogen.


Introduction
Prunus domestica is a flowering plant species belonging to the family Rosaceae.It is commonly known as European plum, common plum, or prune and produces stone fruits, which are typically called plums (Zhebentyayeva et al., 2019).Prusnus domestica is a rich source of vitamins, minerals, organic acids, and fiber, including anthocyanins, flavonol Song et al. 10.3389/fmicb.2024.1458456Frontiers in Microbiology 02 frontiersin.orgderivatives, and phenolic acids (Soares et al., 2023).A number of studies have shown that the consumption of plums promotes a wide range of health benefits; prevents a wide variety of diseases, such as cancer, diabetes, and obesity; has anti-inflammatory properties; improves digestive function; and has significant applications in the fields of medicine and food (Silvan et al., 2020;Abraao et al., 2022;Bahrin et al., 2022;Rybak and Wojdyło, 2023).
Over the past few years, plum-growing areas have increased in Xinjiang, China (Wang et al., 2021).The total plum-growing area (in Chinese pinyin: Xinmei) in Kashgar Jiashi County is more than 28,000 ha, yielding an estimated production volume of 85,000 tons.It accounts for 40% of China's total area of plum production and 60% of China's total plum output (Fan and Zhang, 2023).Due to the gradual expansion of the plum growth area, coupled with mismanagement and inadequate technology, plums are prone to diseases and pests such as spot disease, brown rot, and food worms, which negatively influence their growth.
In the realm of agricultural practices, combating fungal diseases remains a critical concern, particularly in safeguarding the health and yield of essential agricultural and fruit crops.Despite advancements in various agricultural techniques, chemical control continues to stand out as a fundamental tool in the fight against fungal pathogens (Kettles and Luna, 2019).Currently, Mucor species associated with plums were mainly reported from Norway (Børve and Vangdal, 2007), Nigeria (Eseigbe and Bankole, 1996), Pakistan (Hassan et al., 2022), Poland (Tuszyński and Satora, 2003), Saudia Arabia (Gherbawy and Hussein, 2010), South Africa (Kwinda et al., 2015), Turkey (Ghimire et al., 2022), and USA (Hong et al., 2000).Despite the advancements in agricultural practices, the control of Mucor species remains a significant concern for farmers and researchers alike.Plant diseases caused by Mucor species can be prevented and reduced by various management strategies.There have been some reports to control these diseases caused by fungi, such as Mucor species, with fungicides (Koka et al., 2021;Saito et al., 2023), plant extracts (Kinge and Besong, 2021;Jangid and Begum, 2022), and biological control agents (Wallace et al., 2018;Oufensou et al., 2023).The Mucor species associated with plums in China have not been reported.In our investigation of plum diseases and their management in China, a total of 37 Mucor strains were isolated.Based on the morphological and phylogenetic analysis, these isolates were identified as Mucor xinjiangensis sp.nov.A detailed description and illustration are provided for the new species and compared with other closely related taxa.Pathogenicity test confirmed that M. xinjiangensis causative agent of brown rot of plum.Further, 14 fungicides were analyzed to check the inhibitory effect on isolated pathogens.

Sample collection and isolation
Samples were collected during plum disease surveys conducted between 2019 and 2020, from affected orchards in Yingmaili Township (39° 31′ 11″ N 76° 55′ 17″ E), Jiashi County, Kashgar Prefecture, Xinjiang, China.We screened out 15 orchards with disease symptoms, and a total of 28,972 fruits were investigated.A total of 10 trees were observed in each orchard, and a 5-point sampling method was used for the observation (Ni et al., 2008).Disease incidence for each orchard was calculated by observing diseased fruits/total number of fruits observed.A total of 200 diseased fruits were collected and brought to the laboratory.Among them, 70 diseased fruits with typical diseased symptoms were separated for fungal isolation.When conidia were visible, single-spore isolation was performed (Zhang et al., 2013;Brahmanage et al., 2020).Alternatively, diseased tissues were grown on potato dextrose agar (PDA) for tissue isolation, as described by Raza et al. (2019).A total of 37 strains of Mucor species were isolated, and mycelial plugs were stored in 2 mL tubes for long-term storage at 4°C under sterile water.Dry and living cultures were deposited in the Herbarium of Microbiology, Academia Sinica (HMAS), and China General Microbiological Culture Collection Center (CGMCC), respectively.Taxonomic novelty description and nomenclature were deposited in MycoBank.

Culture description
Observations of morphological features were made on 4-to 7-dayold fungal colonies incubated at room temperature (28°C) under nearultraviolet (near-UV) light with 12-h photoperiod and 12-h darkness.A color guide by Kornerup and Wanscher (1967) was used to describe colony color on PDA.The morphological characters were photographed using a Nikon Eclipse Ci-L light microscope (Yokohama, Japan) and an Oplenic D2000 digital camera (USA).Columellae, chlamydospore, sporangia, sporangiophore, and sporangiospores were also observed on slides mounted in 100% lactic acid.

Genomic DNA extraction, polymerase chain reaction amplification, and sequencing
The genomic deoxyribonucleic acid (gDNA) was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1990).Amplification of the internal transcribed spacer (ITS) of isolates was conducted using primer pairs ITS 1 forward (5 TCCGTAGGTGA ACCTGCGG-3) and ITS 4 reverse (5 TCCTCCGCTTATTGATATGC-3) (Khan and Javaid, 2022).An initial basic local alignment search tool (BLASTn) analysis was conducted to screen Mucor species based on their ITS sequences.Mucor strains were also amplified and sequenced for a fragment of 28S rRNA gene with primer pairs NL1 and NL4 (Raza et al., 2019).Each locus was amplified using the polymerase chain reaction (PCR) protocol described by Hurdeal et al. (2021) and Zhao et al. (2023).The PCR reaction was performed in a 25-μL reaction volume using a 15-μL rapid Taq master mix (Vazyme, Nanjing, China), 0.1-mM primers, and 10-ng gDNA.PCR was performed in the following conditions: predenaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C (for ITS) or 56°C (for large subunit [LSU]) for 40 s; extension at 72°C for 45 s, 35 cycles; and elongation at 72°C for 10 min.The PCR products were detected by 1% agarose gel electrophoresis, and sequencing was done by Sangong Bioengineering (Shanghai) Co., Ltd.

Phylogenetic analysis
Phylogenetic relationship and taxonomic distinction for novel species were determined using genetic markers recommended in a recent bibliography of the genus Mucor (Hurdeal et al., 2021;Zhao et al., 2023).A sequence assembly was performed, and necessary corrections were made manually wherever necessary using BioEdit 7.2.5 (Hall, 1999).Bayesian inference (BI) and maximum likelihood (ML) analyses were employed to reconstruct the phylogeny, respectively, with MrBayes 3.2.7 (Ronquist et al., 2012) andRAxML 8.2.10 (Stamatakis, 2014).Based on the Akaike information criterion, MrModeltest 2.3 (Nylander, 2004) was used to estimate the best-fit evolutionary models for the two-locus dataset.The posterior probability (PP) distribution convergence was ensured by running 6,000,000 generations of Markov chain Monte Carlo (MCMC) with a random seed and a stopval = 0.01 MCMC algorithm of four chains.Based on the 50% majority rule and removing the first 25% of the trees sampled, we calculated consensus trees based on the 50% majority rule and PP.It was considered significant if the PP value was greater than 0.95.Selected bootstrap replicates were 1,000, and bootstrap support (BS) ≥70 was considered significant (Li et al., 2023).Sequences generated in this study were deposited in GenBank, and their accession numbers can be found in Table 1.

Pathogenicity test
Two representative strains of Mucor species (CGMCC 3.27539 and 39Z29) were tested for pathogenicity (Hussain et al., 2016).Selected fresh and healthy new plum fruits were surface sterilized with 75% ethanol, and then a 4-mm diameter wound was made in the middle of the fruit using a wound retractor.Mycelial plugs were taken from the margin of the growing colonies of isolates using a 4-mm diameter cork borer.Fresh wounds were inoculated by placing mycelial plugs into the wounds.A plug of PDA with no fungal growth was included as a control.The inoculated fruits were arranged in a sterile humidity (65-70%) chamber for 7 days, and fruits were observed every day.Twenty fruits were inoculated with two representative strains and repeated 3 times.After the incubation period, disease lesions were measured, and pathogens were re-isolated (Song et al., 2020).The morphological characteristics and ITS sequences of the re-isolated fungus were compared to those of the original strains.The inoculation experiment was conducted 2-times to ensure reliability.

Fungicides and adjuvants against pathogen
A total of 14 fungicides, including 10 fungicides and 4 adjuvants (Table 2), were tested in vitro against the pathogen (type strain) (Hussain et al., 2014).The fungicides selected in this study were registered under the Pesticide Inspection Institute, Ministry of Agriculture and Rural Affairs, China. 1 The fungicide solutions were prepared according to the label instructions provided by their manufacturers (Table 2).The recommended concentration was diluted, and 1 mL was added to 49 mL PDA medium and poured into a 9 cm Petri dish.An equal volume of sterile water was added to the control Petri dish.A plug of mycelium (5 mm) was inoculated into a petri dish containing fungicides and kept at 28°C for 7 days with daily checks (Wagner et al., 2019).Each treatment was replicated 4 times.The colony diameter was measured, and the inhibition rate was calculated as follows: Mycelial growth inhibition average growth diameter of con % t trol colonies average growth diameter of treated colonies g / r rowth diameter of control colonies In addition, the average growth rate of mycelium in each treatment was used to screen out fungicides that were effective, and the half maximal effective concentration (EC 50 ) value was calculated to measure the toxicity of the agent, using the recommended concentration as the center (used as control).A total of five concentrations were set with four replicates for each concentration.

Disease symptoms and incidence
The initial symptom of the newly observed disease was small, scattered red spots appearing after the onset of the fruit (Figures 1A-F).It was found that the disease spreads swiftly when the temperature rises, resulting in brown spots that sag and expand around, wrinkle, and harden, and eventually lead to fruit falling from the tree.At high humidity, especially early in the morning, white mycelia were observed on infected fruits.In our investigation, we found that a total of 1,032 fruits were diseased out of 28,972.The disease incidence caused by Mucor species for each orchard was between 0.31 and 7.63% (Figure 1G).

Koch's postulates for pathogenicity test
A total of 70 diseased fruit samples with typical disease symptoms were collected, from which fungi were isolated in 21 samples.This yielded 60 fungal isolates, of which 37 were morphologically identified as Mucor species, representing 61.67% of the total isolates.Plum fruits inoculated with two representative strains showed brown rot symptoms of 1.5-2 cm after 7 days.The symptoms first consisted of brown spots that expanded.As brown spots develop, the infected area becomes wrinkled and then fades to dark brown.Infected fruits were found to have white mycelia.Fruits infected with the representative strains developed similar symptoms to those observed in the field.There were no visible symptoms of brown rot on the non-inoculated control fruit (Figures 1H-K).Strains were recovered from the inoculated diseased fruit symptoms, which were conspecific to the original isolates from the natural diseased fruits, based on microscopic characteristics and 100% similarity in ITS and LSU sequences.

Phylogenetic analyses
Among the total Mucor isolates, five strains were randomly selected basis of different collection sites (fields) and successfully amplified with single ITS fragments.A preliminary comparison of ITS sequences via BLASTn search showed that each strain belongs to Mucor but representative strains (CGMCC 3.27539 and 39Z29) were most closely related to Mucor pseudolusitanicus (MF495059; 96% query cover and 98% identity and MF495059; 98% query cover and 99% identity, respectively).By analyzing the combined dataset of ITS and LSU loci, strains collected from diseased plums were further identified.A total of 93 reference sequences were used, including 91 Mucor species, and two species of the Backusella were used as outgroups, retrieved from GenBank (Table 1).In the concatenated alignment of five Mucor strains and 93 reference taxa, 957 distinct alignment patterns and 29.22% proportion of gaps and completely undetermined characters.Based on the combined ITS and LSU phylogenetic analyses, our strains formed an exclusive and wellsupported clade (93/0.98 for ML/PP) (Figure 2).There was a strong correlation between the topologies of the individual gene trees and the concatenated tree, indicating that both strains recovered from brown spots on plum fruit were distinct species of Mucor.(average = 6.89 ± 1.13 × 4.83 ± 0.63 μm) wide, colorless.Chlamydospore occurring in vegetative hyphae, smooth, thin walled, intercalary, single, in pairs or chains, globose, subglobose, 15-35 × 12-19 μm width (average = 21.45 ± 6.09 × 15.37 ± 1.93 μm).Rhizoids present.Zygospores not observed.
Cultural characteristics: Colonies on PDA are fast growing, reaching 6.8 cm in diameter in 2 days after incubation at 28 ± 1°C, colony medium, slightly raised with an erose edge, rough surface, effuse, well-defined margin; colony from above; dull, medium, whitish to pale yellow, later blackish; from below, pale yellow; not producing pigment in PDA media.Sporulate on PDA.
The effective fungicides and fungicide adjuvants were further tested with different concentrations, including the recommended dosage for virulence determination with the EC 50 value (Figure 5).There are some differences in the effective medium concentration of EC 50 value for the seven agents against the M. xinjiangensis (Figures 6A-H).Among them, difenoconazole showed the best antifungal effects, the highest toxicity, and the lowest EC 50 (0.18 mg/L).In the next two, difenoconazole + azoxystrobin and mancozeb were found to have EC 50 values of 4.21 and 7.16 mg/L, respectively.The antifungal effect of ethylicin was relatively less, with the highest EC 50 value of 167.83 mg/L.The EC 50 value of other fungicide adjuvants ranged from about 13-20 mg/L including prochloraz + iprodione (13.06 mg/L), pyraclostrobin + tebuconazole (15.18 mg/L), and trifloxystrobin + tebuconazole (19.18 mg/L).

Discussion
Prunus domestica is widely grown in high temperature and cool humid regions of northwest China (Cao et al., 2014).An unknown brown rot disease on plum fruit was found in several locations in Xinjiang, and its causal agent was unidentified.In the present study, we identified and described the pathogen as a new species, M. xinjiangensis.In the combined phylogenetic analysis of ITS and LSU, M. xinjiangensis formed a sister clade to M. pseudocircinelloides and M. pseudolusitanicus.Although M. xinjiangensis shares 99% ITS identity with M. pseudocircinelloides (XY07713) and M. pseudolusitanicus (CBS 543.8),LSU shares 100% ITS identity with M. pseudolusitanicus (CBS 540.78), but no LSU blast matches any M. pseudocircinelloides strain.By removing the ambiguous sequences of M. pseudolusitanicus in combined phylogenetic analysis, we found that M. pseudolusitanicus has a wide phylogenetic distribution even within its clade but is different in comparison with its type species (see above notes section).Furthermore, M. xinjiangensis is also genetically close to M. circinelloides and M. ctenidius, which are also pathogenic to plants (Nishijima et al., 2011;Sha and Meng, 2016).Nevertheless, these two species were differentiated from our collection based on morphological characteristics of sporangiophores, sporangia, columellae, and sporangiospores.Mycelial growth inhibition of Mucor xinjiangensis (CGMCC 3.27539) from different fungicides and fungicide adjuvants at different dilution concentrations after 5 days.(A1-A5) Difenoconazole diluted concentration (PDA texture differentiation was caused by fungicide addition) (A1. at 3,000; A2. at 5,000; A3. at 7,000; A4 at 10,000; A5. at 20,000).(B1-B5) Ethylicin diluted concentration (B1. at 2,000; B2. at 3,000; B3. at 4,000; B4 at 5,000; B5. at 6,000).(C1-C5) Mancozeb (C1. at 900; C2. at 1,500; C3. at 3,000; C4 at 4,000; C5. at 6,000).(D1-D5) Difenoconazole + azoxystrobin diluted concentration (D1. at 300; D2. at 600; D3. at 1,500; D4. at 2,400; D5. at 3,000).(E1-E5) Prochloraz + iprodione diluted concentration (E1. at 500; E2. at 1,000; E3. at 2,000; E4. at 4,000; E5. at 8,000).(F1-F5) Pyraclostrobin + tebuconazole diluted concentration (F1. at 2,000; F2. at 6,000; F3. at 8,000; F4 at 10,000; F5. at 15,000).(G1-G5) Trifloxystrobin + tebuconazole diluted concentration (G1. at 1,000; G2. at 2,000; G3. at 4,000; G4. at 8,000; G5. at 10,000). 10.3389/fmicb.2024.1458456 Frontiers in Microbiology 14 frontiersin.orggloeosporioides or Colletotrichum acutatum), respectively (Børve and Vangdal, 2007).Usually, these diseases begin with punctured wounds or insect bites.It is important to note, however, that Mucor rot and Rhizopus rot typically share the same symptoms.Due to Mucor rot or Rhizopus rot, the plums become soft, watery, and covered with black spore masses as the infection develops rapidly (Shahnaz et al., 2021;Seethapathy et al., 2022).In Mucor rot, it is often found that fungal structures are stiffer than those of Rhizopus rot, orientated at specific angles to the fruit surface at the time of maturation (Michalltdes, 1990).Rhizopus fruit rot is usually of less importance than the Mucor brown rot in the field, but both can cause important postharvest losses (Dennis and Mountford, 1975).However, the new brown rot disease reported here is the most severe in the field during June and July under high temperatures following continuous rainfall, when plum  fruit faces continuous sunshine.The effects of sunburn are slow tree decline and browning of the skin on fruit exposed to too much heat (Racsko and Schrader, 2012;Lal and Sahu, 2017).It might be possible that continuous sunshine toward the premature plum promotes the initial infection (red spots), and then airborne Mucor pathogen attacks it and enhances the process of the premature fall of the fruit.Additionally, M. xinjiangensis was isolated from symptomatic plum fruit tissues, which showed different symptoms from Rhizopus rot and abiotic stress (sunburn).Due to these reasons, we propose a new name for the disease, "Mucor brown rot, " in order to distinguish it from two well-known biotic and abiotic diseases.Furthermore, healthy plum fruits inoculated with diseased fruit tissue collected from the field and with an isolated strain (M.xinjiangensis) also showed the same symptoms.
To date, M. xinjiangensis has only been isolated from P. domestica among stone fruits.The inoculation and re-isolation tests confirmed that M. xinjiangensis is pathogenic against P. domestica, and may be even more pathogenic.A range of hosts for M. xinjiangensis is unknown at the moment.Other stone fruits should be investigated for M. xinjiangensis infection.
It was found that different fungicides and fungicide adjuvants have different inhibition effects on pathogenic fungi (Nita et al., 2007).In the present study, 14 fungicides and their combinations were tested and compared with recommended dosage.These fungicides were commonly available in the Xinjiang market to control plant diseases.Among them, the antifungal rate of seven agents was more than 80%.As broad-spectrum low-toxicity, these fungicides are widely used to control fungal diseases on fruit trees, including apple, grape, peach, pear, and plum, in addition to combination formations.They are mainly classified into triazole (Toda et al., 2021), strobilurin (Balba, 2007), imidazole, benzimidazole, dicarboximidie, protection (chlorthalonil, mancozeb), and the plant bionic pesticide ethylicin (Zhang et al., 2020).There is an increasing tendency to combine two fungicides with different mechanisms of action to increase activity and efficacy (Rashid et al., 2014;Cohen et al., 2018) and to delay the emergence of fungicide resistance (Dooley et al., 2015).There are some research reports that explore different classes of fungicides to identify compounds capable of inhibiting Mucor species growth and spore germination.This includes triazoles, benzimidazoles, strobilurins, and other chemical groups commonly used against fungal pathogens (Suárez-García et al., 2021;Yamleshwar and Rai, 2023;Bai et al., 2024).
Our results showed that difenoconazole has the strongest toxicity and the smallest EC 50 .The smaller the EC 50 , the stronger the toxicity of the agent and the better the antifungal effect (Halling-Sørensen, 2000;Hu et al., 2013).This fungicide belongs to the systemic fungicide, which can inhibit the formation of sporangium and prevent the infection of fungi.At the same time, it has a lasting protection and treatment effects, so as to improve crop yield and quality.Zeng et al. (2023) found that thiophanatemethyl, tebuconazole, and difenoconazole showed significant field control effects on pathogens of fruit brown rot on Prunus salicina var.taoxingli.The fungicide test results of our study broaden the control of brown rot disease on plum fruit and the selection of fungicides.Antifungal fungicides were tested for virulence in vitro only, with the EC 50 value serving as a reference value during the in vitro test.Further verification is needed to screen out the effects in the field.Also, we need to examine the application method, the application time, the climate conditions, etc.The sensitivity of pathogenic fungi to fungicides may vary, so screening targeted fungicides is essential (Masiello et al., 2019).Mucor brown rot can be prevented and controlled with difenoconazole, followed by difenoconazole + azoxystrobin and mancozeb as alternatives or rotation agents, in order to prevent the development of pathogen resistance due to long-term or repeated use of the same fungicide.
Each measurement was repeated at least 3 times.Dunnett tests were used to compare mean values based on univariate analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS) version 19.0 (IBM) software.Different letters above the bars indicate statistical differences (p < 0.05).

FIGURE 1
FIGURE 1 Disease development in the field on plum tree and pathogenicity test.(A) Infected plant.(B−F) Gradually disease expansion on fruit collected from different orchards.(G) Disease incidence in 15 orchards.(H) Control.(I) Inoculated with Mucor xinjiangensis, strain CGMCC 3.27539.(J) Inoculated with M. xinjiangensis, strain 39Z29.(K) Inoculated directly with diseased plum tissue collected from the field.

TABLE 1
Reference specimens and their GenBank accession numbers were used for phylogenetic analysis in this study.

TABLE 2
Fungicides and fungicides/adjuvants mixture tested against Mucor xinjiangensis in this study.