Citrus melanose, caused by Diaporthe citri, is one of the most serious diseases in citrus causing a damage rate of more than 95% when there were no applications of control measures (Hyun et al., 2004; Koh et al., 1996). Most farms protect the melanose by a chemical control measure, spraying chemicals 4-5 times from June to August including the rainy season from late June to mid-July a crucial period of more than 70% of disease infections (Hyeon, 2019). The frequent fungicide application may increase the risk of developing resistant pathogens (Aktar et al., 2009). Indeed, in the previous study isolate D. citri NEL21-2 was found as a resistant to systematic fungicide benomyl, whose hyphae grew well even at the highest chemical concentration. Furthermore, abundant isolates were regarded as resistant to kresoxim-methyl, which could not inhibit the mycelium growth by more than 50% (Soe et al., 2023a).
In the life cycle of many fungi, the asexual spore is critical due to the primary means for dispersion and serves as a ‘safe house’ for the fungal genome in adverse environmental conditions (Osherov and May, 2001). Also, there is well-known knowledge that inhibition of hyphal growth is a major way for disease suppression in many crop plants. There are many studies that revealed the infection structures on the resistant host leaves in which the fungal conidia didn’t geminate or hyphae growth was inhibited (An et al., 2010; Kim and Jeun, 2007). The infection structure of resistant isolates expected to be similar with those observed on resistant plants. In this study, the infection structures were observed on both fungicides treated and untreated parts of citrus leave after inoculation with susceptible and resistant isolates.
Benomyl resistance is common in fungi, especially after multiple applications (Albertini et al., 1999; Cooley and Caten, 1993; Koenraadt et al., 1992; Ma et al., 2003; Yarden and Katan, 1993). Fungal resistance to benomyl is typically associated with point mutations in a β-tubulin gene that changes the amino acid sequence at the benzimidazole binding site (Koenraadt et al., 1992). The site of amino acid of β-tubulin gene causing resistant mutant are different by the fungal pathogens. Some pathogens show resistance due to a single amino acid change of β-tubulin gene, while others have two or more changes (Ma and Michailides, 2005). For example, two β-tubulin genes have been described for some Colletotrichum spp. (Buhr and Dickman, 1994; Jayasinghe and Fernando, 2000; Panaccione and Hanau, 1990; Peres et al., 2004). In C. graminicola infecting maize and in C. gloeosporioides f. sp. aeschynomene affecting Northern jointvetch (Aeschynomene virginica (L.)), two genes, TUB1 and TUB2, have only 73% and 69% sequence identity, respectively (Buhr and Dickman, 1994; Panaccione and Hanau, 1990). A point mutation in TUB2 that results in a substitution of a glutamic acid for a lysine at amino acid 198 confers benomyl resistance in both species (Buhr and Dickman, 1994; Panaccione and Hanau, 1990).
Like all strobilurins, kresoxim-methyl acts as a specific inhibitor of respiration by binding to the center Qp of cytochrome b (Anke, 1995; Becker et al., 1981; Clough et al., 1995; Mizutani et al., 1995, 1996a; Sauter et al., 1995; Von Jagow and Link, 1986). The site-specific mode of action of Strobilurin fungicides imposes a potential risk of resistance development (Köller, 1991). In addition to target site mutations, Strobilurin sensitivities can also be diminished by the induction of alternative respiration in response to the action of respiration inhibitors such as Strobilurins (Day et al., 1995). This alternative pathway, which is active in the presence of alternative oxidase (Li et al., 1996), has been identified as the cause for relatively low Strobilurin sensitivities of fungal mycelia (Hayashi et al., 1996; Mizutani et al., 1995, 1996a, 1996b; Olaya et al., 1998; Shirane et al., 1995). In several Saccharomyces cerevisiae mutants, substitutions of amino acids within the Strobilurin binding site of cytochrome b were found to decrease Strobilurin sensitivities (Di Rago et al., 1989; Geier et al., 1992, 1994). A similar mechanism was reported for fungal organisms naturally tolerant to Strobilurins, such as the Strobilurin-producing basidiomycetes Strobilurus tenacellus and Mycena galopoda, and for the fission yeast Schizosaccharomyces pombe (Kraiczy et al., 1996). Thus far, a similar mechanism of mutational Strobilurin resistance or natural tolerance has not been reported for any plant-pathogenic fungus. For Venturia inaequalis, the amino acid sequence determined for the cytochrome b target site indicated full sensitivity to Strobilurins (Zheng and Köller, 1997). Also, in Alternaria solani or Pyrenophora teres the mutations F129L, G137R and G143A amino acids of cytochrome b have been reported (Semar et al., 2007).
Therefore, in this study, besides the observation of infection structure, in order to illustrate the mechanism of chemical resistance against benomyl or kresoxim-methyl, either genome sequences of β-tubulin for benomyl or that of cytochrome b for kresoxim-methyl was analyzed with the resistant isolates of D. citri.
Materials and Methods
Fungal isolates
Resistant NEL7-2 and susceptible NEB7-3 isolates against kresoxim-methyl and resistant NEL21-2 and susceptible NEL6-1 isolates against benomyl from the previous study of Soe et al. (2023a) were selected as representative chemical-resistant and susceptible D. citri isolates for observation of the infection behavior at 1, 2, and 3 days after inoculation on the inoculated citrus leaves.
Inoculum production
Mycelium tips of resistant NEL21-2 and NEL7-2 as well as susceptible NEL6-1 and NEB7-3 of D. citri isolates growing on potato dextrose agar medium (Becton, Dickinson, and Company, Claix, France) were sub-cultured on fresh oatmeal agar medium (Becton, Dickinson, and Company). To achieve sporulation, the fungi were incubated under fluorescence light at 25°C for 4 weeks in an incubator (DA MIL-2500, Dong-A, Siheung, Korea).
Fungicide treatment
Young citrus leaves, which were not hardened, were soaked in 1% sodium hypochlorite solution (NaClO) for 30 s, followed by 70% ethanol for 30 s, then rinsed three times in sterile distilled water for 30 s. These leaves were laid in a Petri dish (ø = 90 mm) with sterilized filter paper. The end of the petiole was wrapped with cotton wool soaked with sterilized water to keep the turgor pressure of the leaves. These citrus leaves were pre-sprayed with kresoxim-methyl at 0.5 ml/l or benomyl at 0.65 g/l until the leaves were completely wet. The pre-treated leaves were kept for 3 h at room temperature until the moisture of the leaves dried and used for fungal inoculation. The leaves were sprayed with H2O instead of the chemicals as a negative control.
Fungal inoculation
A couple of sets of resistant NEL7-2 and susceptible NEB7-3 isolates for kresoxim-methyl and in a different combination of resistant NEL21-2 and susceptible NEL6-1 isolates for benomyl were used as inoculum. To establish an inoculation solution, 10 ml of distilled water was added to oatmeal agar medium formed with pycnidia of the fungus. Conidia were harvested using a loop and filtered with a double folded mira-cloth (475855-1R, Merck KGaA, Darmstadt, Germany). The concentration of inoculum was adjusted to 1 × 106 conidia/ml using a hemocytometer (Hausser Scientific Inc., Horsham, PA, USA). After adding 0.01% Tween 20 (BIOSESANG Co., Ltd., Yongin, Korea), 20 μl of the inoculum was dropped onto citrus leaves. On the left side of the leaves by resistant isolates and on the right side of the leaves by susceptible isolates were inoculated. The inoculated leaves were kept in an incubator (DA MIL-2500, Dong-A) at 25°C until the symptom was visible. The experiments were temporally separated into three replications and every treatment contained three inoculation sites in one leave.
Fluorescence microscopy
To unveil the resistance phenomenon on the citrus leaves, both treated with chemicals and untreated leaves were observed with a fluorescent microscope (BX60, Olympus, Tokyo, Japan) at 1, 2, and 3 days after inoculation. Inoculated sites of the leaves were cut with a razor blade and fixed with 2% glutaraldehyde in phosphate buffer (pH 7.2) at 4°C for 2 h. The primary fixed samples were washed with the phosphate buffer three times (10 min each) and dyed with 0.2% diethanol (UVtex-2B, Fungiqual A, Muellheim, Germany) for 40 m at room temperature. These samples were washed with the phosphate buffer 4 times again (10 min each) and mounted with 50% glycerin on glass slides. Infected sites on the samples were observed with the fluorescent microscope equipped with a filter set (exciter filter, BP 400-440; interference beam splitter, FT 460; barrier filter, LP 470). Germination rate (%) and lengths of germ tube (μm) of the fungal isolates were determined for all treatment groups. More than 100 conidia were randomly observed at one inoculation site, each. The germination rate (%) was calculated as [(number of germinated conidia)/(total number of conidia)] × 100. The lengths of germ tube (μm) were measured with Image J software (https://imagej.en.softonic.com/ij153-win-java8). The experiments were temporally separated into three replications with each of the three samples.
Analysis of β-tubulin and cytochrome-b DNA sequences
To illustrate the resistance mechanism sequences of two genes β-tubulin and cytochrome b were analyzed which has been known as an inhibitor of binding sites of bezimidazol and quinone outside inhibitor (QoI), respectively. The primers for β-tubulin gene sequences were DCBeta-F (5′-GAC CCG ACT CGA CAA TAA CG-3′) and DCBeta-R (5′-ATT CAC TCC TCG CCC TCA AG-3′) which were designed using a clone manager program (version 9.0). The condition of PCR was at 94°C for 5 min, 30 steps for 94°C 1 min, 58.2°C 30 s, and 72°C for 2 min and final extension at 72°C for 7 min. The primers for cytochrome b were Cyto129-F (5′-TTA CAC GCT AAC ACT GCT TC-3′), Cyto129-R (5′-ACA CCA TGC TTC TTT ACT G-3′) for 129 and 137 amino acids and for 143 amino acids the primers of Cyto143-F (5′-GAC AGG CTG GGT CAC TAA TG-3′), Cyto143-R (5′-CCT CCG GGT GGT TAA GAA G-3′) were designed. PCR condition was the same with that of β-tubulin except the annealing temperatures were 51.5°C and 57.3°C, respectively.
The amplified PCR products were carried out sequencing service from a biotechnology company (Macrogen, Seoul, Korea). Sequencing data of both forward and reversed primer were aligned using genetic analysis program MEGA (version 11.0.13).
Phylogenic analysis of D. citri isolates
To investigate whether genetic differences of the resistant isolates compared to the susceptible, the DNA sequences of rDNA internal transcribed spacer (ITS) region were analyzed. Total 111 isolates of D. citri were obtained from Jeju Island according to the method from the previous study of Soe et al. (2023b). Genomic DNA was extracted with phenol-chloroform extraction assays from Aamir et al. (2015). PCR amplification of ITS region of D. citri isolates was carried out according to the previous methods (Soe et al., 2023b). Phylogenetic analysis of D. citri isolates was performed using partial ITS gene sequences. All ITS1 and ITS4 sequences of D. citri isolates were manually adjusted as the first alignments for assembled sequences by using the BioEdit Sequence Alignment Editor (BioEdit v7.2.5). These manually aligned sequences were used in the program of Molecular Evolutionary Genetics Analysis version 11 (MEGA11). Phylogenetic analysis was undertaken using the neighbor-joining method and bootstrap support values of 1,000 replicates were used.
Results
Symptoms on the inoculated citrus leaves
The leaves untreated by both chemicals showed apparent melanose lesions by inoculation with both resistant and susceptible isolates at 5 days after inoculation (Fig. 1A and C). However, on the chemical pre-treated leaves, no apparent symptom appeared after inoculation with susceptible isolates such as NEL6-1 and NEB7-3, which are susceptible against benomyl and kresoxim-methyl, respectively (Fig. 1B and D, right side). Remarkably, melanose symptoms were shown even on both chemical pre-treated leaves after inoculation with the resistant isolates, NEL21-2 against benomyl and NEL 7-2 against kresoxim-methyl, respectively (Fig. 1B and D, left side).
Conidial germination rate of D. citri
On the chemically unsprayed leaves, most conidia started to germinate at 1 day after inoculation with all isolates of D. citri (Figs. 2A and D, 3A and D). Two days after inoculation, the majority of the germ tube began to lengthen with both isolates of D. citri (Figs. 2B and E, 3B and E). The fast growth of mycelium with both isolates of D. citri was observed at 3 days after inoculation (Figs. 2C and F, 3C and F). Also, there was no statistically significant difference in the conidial germination rates at 1, 2, and 3 days after inoculation between the resistant and susceptible isolates (Fig. 4A and C). It indicated that there are no differences in pathogenesis between the resistant and the susceptible isolates without any fungicides.
However, on the leaves pre-treated with benomyl, most conidia of resistant isolate NEL21-2 germinated similar to those on the untreated leaves at 1, 2, and 3 days after inoculation (Fig. 2G-I), whereas the hyphae growth of susceptible isolate NEL6-1 was strongly limited all time long after inoculation (Fig. 2J-L). These different germination rates were statistically significant between the resistant and susceptible isolates on the benomyl pre-treated leaves (Fig. 4B).
Like on the benomyl pre-treated leaves, kresoxim-methyl could not suppress the conidial germination of the resistant isolate NEL7-2 (Fig. 3G-I), whereas the abundant conidia of the susceptible isolate NEB7-3 were not germinated by the treatment with kresoxim-methyl (Fig. 3J-L). The difference in germination rate between the resistant and susceptible isolates was significant on the kresoxim-methyl pre-treated leaves (Fig. 4D).
Hyphae growth of D. citri
All isolates of D. citri developed quickly to generate hyphae 3 days after inoculation on untreated citrus leaves (Figs. 2C and F, 3C and F). Also, there was no statistically significant difference in the hyphal growth at 3 days after inoculation (Fig. 5A and C) between the resistant and susceptible isolates.
However, on the leaves pre-treated with benomyl, the hyphae length of resistant isolate NEL21-2 was observed to be similar to that of the untreated leaves at 3 days after inoculation (Fig. 2I), whereas not only the elongation of germ tubes but also the hyphae growth of susceptible isolate NEL6-1 was strongly limited (Fig. 2L). These different hyphae lengths were statistically significant between the resistant and susceptible isolates on the benomyl-pre-treated leaves (Fig. 5B).
Similar on the benomyl pre-treated leaves, kresoxim-methyl showed the hyphae length of the resistant isolate NEL7-2 was similar to that on the untreated leaves (Fig. 3I), whereas the elongation rate of germ tubes and growth of hyphae of the susceptible isolate NEB7-3 were suppressed (Fig. 3L). The difference in hyphae length between the resistant and susceptible isolates was significant on the kresoxim-methyl pre-treated leaves (Fig. 5D).
Analysis of β-tubulin and cytochrome b DNA sequences
Sequencing data of β-tubulin was compared between resistance and susceptible isolates against benomyl. The 198th amino acid of resistance isolate NEL21-2 was shown to encoding alanine, whereas most susceptible isolates including the strain from NCBI encoded glutamic acid (Fig. 6). It indicated that a mutation has occurred at the 198th amino acid in the β-tubulin gene which may play a role to expression of resistant against Benomyl.
However, no point mutation in the resistant isolates against kresoxim-methyl was found in 129th, 137th, and 143th amino acids of the cytochrome b gene, which has changed its DNA sequence in the resistant strains of other fungal pathogens (Supplementary Fig. 1).
Phylogenic analysis of D. citri isolates
Phylogenetic trees inferred from rDNA ITS sequences. It has been found that some resistant isolates of D. citri against kresoxim-metyl have genetic homology with the susceptible isolates. However, most of resistant isolates formed one large clade which was distinct from the clades of susceptible isolates (Fig. 7). It indicated that a genetical change may occur in the resistant isolates because of exterior environment such as often exposure with agrochemicals. Also, the resistant isolate NEL21-2 against benomyl belonged to the same clade with resistant against kresoxim-methyl.
Discussion
To compare the infection behavior between the resistant and susceptible D. citri isolates against the benomyl or kresoxim-methyl, fluorescent microscopic observations on the surfaces of citrus leaves were carried out. In the previous study of Soe et al. (2023a), one isolate NEL21-2 showed resistance against benomyl whose mycelia growth wasn’t inhibited even at the highest concentration of benomyl. It works by disrupting tubulin polymerization, which inhibits fungal growth (Zhou et al., 2016). The fluorescent microscopic observations showing no decrease in conidial germination rate (Fig. 4B) and hyphae growth (Fig. 5B) of the isolate NEL21-2 was coincide with the previous study. By these observations, it was unveiled the resistant expression of the resistant isolate NEL21-2 against benomyl, i.e., no suppression efficacy by the fungicide on germination or hyphal growth of the isolate.
More isolates were resistant against kresoxim-methyl than in the case of benomyl, in which only one resistant isolate was found. Like the case of benomyl, kresoxim-methyl could not suppress the conidial germination rate and hyphae growth of the resistant isolate NEL7-2 on the fungicide pre-treated leaves (Figs. 4D and 5D). These results indicated that the resistant isolates could overcome the fungicide mechanism which may suppress the fungal germination or hyphal growth of susceptible isolates.
Kresoxim-methyl significantly suppressed the infection compared to that of the control detached citrus fruit (Liu et al., 2023). However, there have been also reported some resistant pathogenic fungi against QoI fungicide. Kresoxim-methyl could not inhibit the conidial germination of resistant isolates of V. inaequalis even at the higher concentration than these considered as resistant (0.1 μg/ml) (Sallato et al., 2006). Moreover, other QoI fungicides such as azoxystrobin and pyraclostrobin failed to inhibit spore germination, germ tube length, and mycelium growth of resistant isolates of Colletotrichum acutatum (Forcelini et al., 2016).
Chemical resistance could be explained by mutation of the pathogen. In case of benzimidazole fungicides, resistance had mostly occurred through point mutations in β-tubulin gene resulting in change of the benzimidazole binding site (Ma and Michailides, 2005). Affection of β-tubulin gene to benzimidazole resistance was reported in several fungal pathogens such as Botrytis cinerea, Cercospora beticola, Gibberella zeae, Neurospora crassa, Tapesia yallundae, Tapesia acuformis, and V. inaequalis which was shown high resistant against benomyl by β-tubulin at 198th amino acid point mutation (Albertini et al., 1999; Chen et al., 2009; Koenraadt et al., 1992; Trkulja et al., 2013; Yarden and Katan, 1993). In our research, the NEL21-2 strain, which showed the highest resistance to benomyl, revealed a point mutation at the 198th amino acid of β-tubulin. These results indicate that the 198th amino acid of β-tubulin plays a crucial role in conferring high resistance to benomyl in D. citri.
Similarly, cytochrome b has been known as an inhibitor of QoI fungicide binding site (Anke, 1995; Baldwin et al., 1996; Clough et al., 1995; Mizutani et al., 1995, 1996a; Sauter et al., 1995; Von Jagow and Link, 1986). Sequencing of the cytochrome b gene showed that QoI-resistant isolates contained either G143A, G137R, or F129L amino acid substitutions (Semar et al., 2007). In stored mandarin fruit, B. cinerea has achieved resistance against azoxystrobin in which the G143A point mutation in the cytochrome b gene was examined (Saito and Xiao, 2018). However, in this study, no point mutation at any amino acid sites was found in the resistant isolates of D. citri (Supplementary Fig. 1). Not all resistant pathogens have the point mutation found in cytochrome b gene. For example, some resistant Colletotrichum gloeosporioides species against Strobilurin fungicides did not correspond with mutations in the cytochrome b gene (Chu et al., 2022). Probably, the resistance of D. citri to kresoxim-methyl may be also caused by other mechanisms rather than mutation in cytochrome b gene.
Some plant pathogens have not developed QoI resistance even under the exposure of frequent application of the fungicide (Koch et al., 2008; Semar et al., 2007; Sierotzki et al., 2007). The reason for this has not been clearly identified which may be a very complex genetic outcome. For example, it has been reported that in the case of Monilinia, resistance occurrence might be suppressed by the insertion of intron after the 143rd amino acid of cytochrome b gene (Miessner and Stammler, 2010). Therefore, it can be predicted that in the case of D. citri, resistance may have occurred due to genetic variations other than cytochrome b, which has not been clearly unveiled, yet. The different ITS sequences between susceptible and resistant isolates of D. citri (Fig. 6) may be not enough to explain the occurrence of resistant isolates.
In conclusion, strong infection was observed by the resistant isolates even after pre-treatment either benomyl or kresoxim-methyl. In the resistant isolates, conidia could germinate, and hyphae grew without any suppression effect by both fungicides. These infection structures of the resistant isolates may be caused by a point mutation or genomic change of intron. This study may help to understand the resistant expression of D.citri isolates against fungicides.
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