Phylogenetic and Pathogenic Evidence Reveals Novel Host–Pathogen Interactions between Species of Lasiodiplodia and Citrus latifolia Dieback Disease in Southern Mexico

Mexico ranks second in the world for Persian lime (Citrus latifolia) exports, making it the principal citrus exporter within the national citrus industry, exporting over 600,000 tons per year. However, diseases are the main factor reducing production, resulting in significant economic losses. Among these diseases, fungal diseases like dieback, caused by species of Lasiodiplodia, are an emerging issue in Persian lime. Symptoms include gummosis, twig and branch dieback, cankers, the necrosis of bark and wood, fruit mummification, and tree decline. The aim of this study was to investigate the occurrence and pathogenicity of the fungal species associated with twig and branch dieback, cankers, and decline of Persian lime trees in southern Mexico, and to elucidate the current status of the Lasiodiplodia species causing the disease in Mexico. During June, July, and August of 2023, a total of the 9229 Persian lime trees were inspected across 230 hectares of Persian lime orchards in southern Mexico, and symptoms of the disease were detected in 48.78% of the trees. Branches from 30 of these Persian lime trees were collected. Fungal isolates were obtained, resulting in a collection of 40 strains. The isolates were characterized molecularly and phylogenetically through the partial regions of four loci: the internal transcribed spacer region (ITS), the β-tubulin gene (tub2), the translation elongation factor 1-alpha gene (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2). Additionally, pathogenicity was assessed, successfully completing Koch’s postulates on both detached Persian lime branches and certified 18-month-old Persian lime plants. Through multilocus molecular phylogenetic identification, pathogenicity, and virulence tests, five species were identified as causal agents: L. iraniensis, L. lignicola, L. mexicanensis, L. pseudotheobromae, and L. theobromae. This study demonstrates that in southern Mexico, at least five species of the genus Lasiodiplodia are responsible for dieback in Persian lime. Additionally, this is the first report of L. lignicola and L. mexicanensis as causal agents of the disease in citrus, indicating novel host interactions between species of Lasiodiplodia and C. latifolia.


Introduction
Globally, citriculture is regarded the most important agricultural endeavor within fruit production, yielding over 150 million tons annually.Citrus fruits are cultivated on all five aurantifolia, and C. sinensis were L. hormozganensis, and L. theobromae; in C. reticulata, it was L. iraniensis [26]; and, in the United States of America, L. iraniensis, and L. parva have been reported as causing gummosis and dieback in C. sinensis, and Citrus sp., respectively [13,27].
Worldwide, the studies on dieback caused by Lasiodiplodia in citrus have not focused on Persian lime.Only one study in Mexico found that the disease agents were L. brasiliense, L. citricola, L. pseudotheobromae, L. subglobosa, and L. theobromae [28].However, this study used only the ITS, tef-1α, and tub2 regions.Currently, for species of the genus Lasiodiplodia, the use of ITS, tef-1α, tub2, and rpb2 results in a more reliable species-level resolution [10,15].Therefore, some identification errors of the species reported for Persian lime might have occurred.
In the past five years, dieback of Persian lime caused by Lasiodiplodia has not been studied.Therefore, it is important to understand the current status of Lasiodiplodia species causing dieback in Persian lime.Moreover, in southern Mexico, specifically in the state of Tabasco, the etiological agents have not been determined; this state contributes over 87 thousand tons to Persian lime production [2].The objectives of this study were to (i) identify Lasiodiplodia species associated with dieback of Persian lime in southern Mexico, (ii) compare the previously described species associated with dieback in Persian lime with the current state of the species in the region, using the four recommended molecular markers, and (iii) evaluate their pathogenicity and virulence in excised green shoots and certified nursery plants of Persian lime.

Field Survey and Sampling
During June, July, and August of 2023, a survey was conducted of 230 hectares of Persian lime in the main producing region of Tabasco, Mexico.A total of thirty symptomatic plant tissues showing canker, gummosis, and branch dieback were collected (Figure 1), utilizing a completely random sampling method for symptomatic trees.The plant tissue was stored in marked plastic bags and placed in a plastic container with ice for transport to the laboratory.
theobromae have been identified in citrus branches (Citrus sp. and C. aurantifolia) as causing cankers and dieback symptoms [23,24]; in Mexico, L. theobromae was reported to cause dieback of C. sinensis [25].In Oman, the causal agents of dieback and gummosis in C. aurantifolia, and C. sinensis were L. hormozganensis, and L. theobromae; in C. reticulata, it was L. iraniensis [26]; and, in the United States of America, L. iraniensis, and L. parva have been reported as causing gummosis and dieback in C. sinensis, and Citrus sp., respectively [13,27].
Worldwide, the studies on dieback caused by Lasiodiplodia in citrus have not focused on Persian lime.Only one study in Mexico found that the disease agents were L. brasiliense, L. citricola, L. pseudotheobromae, L. subglobosa, and L. theobromae [28].However, this study used only the ITS, tef-1α, and tub2 regions.Currently, for species of the genus Lasiodiplodia, the use of ITS, tef-1α, tub2, and rpb2 results in a more reliable species-level resolution [10,15].Therefore, some identification errors of the species reported for Persian lime might have occurred.
In the past five years, dieback of Persian lime caused by Lasiodiplodia has not been studied.Therefore, it is important to understand the current status of Lasiodiplodia species causing dieback in Persian lime.Moreover, in southern Mexico, specifically in the state of Tabasco, the etiological agents have not been determined; this state contributes over 87 thousand tons to Persian lime production [2].The objectives of this study were to (i) identify Lasiodiplodia species associated with dieback of Persian lime in southern Mexico, (ii) compare the previously described species associated with dieback in Persian lime with the current state of the species in the region, using the four recommended molecular markers, and (iii) evaluate their pathogenicity and virulence in excised green shoots and certified nursery plants of Persian lime.

Field Survey and Sampling
During June, July, and August of 2023, a survey was conducted of 230 hectares of Persian lime in the main producing region of Tabasco, Mexico.A total of thirty symptomatic plant tissues showing canker, gummosis, and branch dieback were collected (Figure 1), utilizing a completely random sampling method for symptomatic trees.The plant tissue was stored in marked plastic bags and placed in a plastic container with ice for transport to the laboratory.

Fungal Isolations from Persian Lime Branches with Dieback
Fungal isolates were obtained following standard protocol [28].Fragments of approximately 3 cm from each branch were cut from the margin between the necrotic and healthy tissue zones.These were placed into a 50 mL conical tube containing 20 mL of water plus 5% commercial powdered detergent for 10 min to remove dirt and insects, then immersed in 0.6% sodium hypochlorite for 1 min, rinsed three times with sterile water, and blotted dried on sterile paper.Five pieces of wood (approximately 5 mm 2 each) were placed into 100-by-15 mm Petri dishes containing potato dextrose agar (PDA; Difco, Detroit, MI, USA, 49 g L −1 ) supplemented with 0.5 g L −1 streptomycin sulfate and 0.4 g L −1 penicillin (Sigma-Aldrich Co., St. Louis, MO, USA).Plates were incubated at 25 • C for 48 h in the dark.
Selected emerging fungal colonies were transferred to Petri dishes containing 2% water agar, incubated in the dark for 48 h, and purified by transferring hyphal tips to Petri dishes containing PDA and incubated at 25

DNA Extraction, Polymerase Chain Reaction Amplification, and Sequencing
Isolates were cultured on PDA and incubated at 25 • C for 7 days.Aerial mycelium was directly collected from the medium using a sterile scalpel blade and transferred into 2 mL microtubes.Total genomic DNA was extracted using the cetyl trimethylammonium bromide (CTAB) method with slight modifications [29].DNA concentrations were quantified using a NanoDrop OneC (Thermo Fisher Scientific, Madison, WI, USA), the DNA samples were diluted to a concentration of 100 ng/µL.Partial regions of four loci, the internal transcribed spacer region (ITS), the β-tubulin gene (tub2), the translation elongation factor 1-alpha gene (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2) were amplified using specific primer sets (Table 1).All amplification reactions were performed in a total 25 µL volume mixture consisting of 12.5 µL of BlasTaq 2X PCR MasterMix (Applied Biological Materials, Vancouver, BC, Canada), 9.5 µL of Water Molecular Biology, 1 µL of each forward and reverse primer at a concentration of 10 µM, and 1 µL of 100 ng/µL DNA template.The amplification conditions comprised an initial denaturation step at 95 • C for 3 min, followed by 35 cycles of denaturation at 95 • C for 15 s, annealing at 55 • C for ITS region, 58 • C for tef1-α gene, and 60 • C for tub2 and rpb2 genes for 15 s, and extension at 72 • C for 10 s, followed by a final extension at 72 • C for 5 min.The PCR assays were conducted in a MiniAmp plus thermocycler (Thermo Fisher Scientific, Madison, WI, USA).The PCR products were separated by electrophoresis in a 1.5% agarose gel at 60 V for 90 min stained with ethidium bromide.The amplified PCR products were purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced in both directions by LANBAMA Laboratory (IPICYT, SLP, San Luis Potosi, Mexico), using the Sanger method.

Phylogenetic Analyses
Forward and reverse sequences were assembled using the Staden Package [34].Sequences of each of the ITS, tef1-α, tub2, and rpb2 loci from 36 well-documented extype Lasiodiplodia species from culture [15] were retrieved from GenBank and aligned with sequences of the isolates obtained in this study (Table 2) using the MAFFT v.7 sequence alignment program [35].The alignments were then manually checked and edited using MEGA XI [36].Subsequently, the alignment of each locus was loaded into SequenceMatrix v.1.8[37] to construct the concatenated matrix.The phylogenetic trees for each locus (ITS, tef1-α, tub2, and rpb2) and for the concatenated matrix were inferred using both maximum likelihood (ML) and Bayesian inference (BI) criteria.ModelTest-NG v.0.1.7 [38] was employed to select evolutionary models independently for each locus and for all loci under the Akaike information criterion (AIC) in both BI and ML analyses.
ML analyses were performed using RAxML-HPC2 [39], with nonparametric bootstrap iterations run for 1000 replications employing the GTR+G+I substitution model.BI was conducted using MrBayes on XSEDE (v.3.2.7a) [40], implemented on the CIPRES Science Gateway portal (www.phylo.org,accessed on 1 July 2024) [41].The BI trees were constructed utilizing the Markov chain Monte Carlo (MCMC) algorithm with four runs and four chains per run, running 10,000,000 generations.Trees and parameter values were sampled every 1000 generations, resulting in 10,000 trees.The initial 2500 trees were discarded as the burn-in phase, and the remaining 7500 trees were used to calculate the posterior probabilities (PPs) in the majority rule consensus tree.Tree topologies were visualized using the FigTree v1.4.0 program [42].Sequences generated in this study were deposited in GenBank (Table 2), and the alignments and trees are available from TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S31354,accessed on 1 July 2024).

Pathogenicity Tests on Detached Branches of Persian Lime
The pathogenicity of the fungal strains was evaluated based on their ability to induce necrosis and gummosis in detached shoots collected from symptomless C. latifolia trees, following the methods outlined by Adesemoye et al. (2014) and Berraf-Tebbal et al. (2020) [19,27].Shoots with a diameter of 15 mm and approximately 20 cm in length were se-lected.They were then surface-disinfected with water containing 5% commercial powdered detergent for 10 min to remove dirt and insects, followed by treatment with 70% ethanol.Subsequently, the shoots were wounded on an intermediate internode using a scalpel.
For each strain, a 5 mm diameter mycelial disk taken from a 7-day-old colony growing on PDA was placed into the wound.Negative controls were inoculated with fresh, noncolonized PDA plugs.The point of inoculation was covered with parafilm to prevent desiccation.The detached branches were then well watered and maintained in a humid chamber under laboratory conditions.Three replicates per isolate were used, and an equal number of detached branches served as controls.One month after inoculation, the lengths of lesions produced by each strain were measured.Necrotic tissue from the margin of the lesions was collected at 30 days after inoculation, placed onto PDA, and molecularly identified to fulfill Koch's postulates.

Pathogenicity on Persian Lime Plants from Certified Commercial Nursery and Virulence Tests
The pathogenicity of the 12 representative Lasiodiplodia isolates identified phylogenetically was tested on healthy 18-month-old Persian lime plants obtained from a certified commercial nursery.The inoculation procedure followed the protocol described by Bautista-Cruz et al. (2019).Each Persian lime plant was wounded 30 cm from the grafting area using a sterile scalpel, and a colonized PDA disk (5 mm diameter) from a 7-day-old culture was placed onto the wound site.The inoculation site was then covered with wet sterile cotton and sealed with parafilm to prevent desiccation.Five plants were inoculated with each isolate, while the control group received noncolonized PDA disks.Immediately after inoculation, each plant was enclosed in a plastic bag sprinkled with sterile distilled water for 72 h to maintain humidity.All plants were kept in a greenhouse under natural light and temperature conditions [28].
Virulence assessments were conducted 30 days after inoculation by removing the bark and measuring the lesion length in the wood using a digital caliper.The experiment was conducted twice to ensure accuracy and reliability.In both experiments, differences in virulence among Lasiodiplodia strains were analyzed with a one-way ANOVA and using the minimum significant difference (p ≤ 0.05) test with R v.3.5.1 statistical software.
To complete Koch's postulates in both experiments, necrotic tissue from the margin of the lesions was sampled and plated onto PDA.The recovered fungal isolates were identified by amplifying and sequencing the tef1-α region.Since control plants did not display necrosis, cankers, or gummosis symptoms, and Lasiodiplodia spp.were not recovered from the mock-inoculated negative controls, it can be inferred that the plants were not latently infected with these pathogens prior to inoculation.

Sample Collection, Isolation, and DNA Sequencing
Out of the 9229 Persian lime trees inspected across 230 hectares of Persian lime orchards in the state of Tabasco, Mexico, symptoms of gummosis, stem cankers, twig and branch dieback, fruit mummification, and decline (Figure 1) were detected in 4502 trees, representing a disease incidence of 48.78%.A total of 40 fungal isolates were obtained from diseased tissues collected from symptomatic Persian lime trees; cultural variability was observed in terms of the growth and color of each strain (Figure S1).
The 40 fungal strains obtained from Persian lime plants exhibiting cankers, as well as twig and branch dieback symptoms were identified at the genus level based on BLAST analysis of the ITS region, with 28 identified as Lasiodiplodia spp.additionally, their cultural characteristics of growth on PDA were also consistent with those of the Lasiodiplodia genus (Figure 2).
was observed in terms of the growth and color of each strain (Figure S1).
The 40 fungal strains obtained from Persian lime plants exhibiting cankers, as well as twig and branch dieback symptoms were identified at the genus level based on BLAST analysis of the ITS region, with 28 identified as Lasiodiplodia spp.additionally, their cultural characteristics of growth on PDA were also consistent with those of the Lasiodiplodia genus (Figure 2).The other 12 isolates belonged to the genera Diaporthe (3), Fusarium (8), and Pestalotiopsis (1) (Figure S1).Derived from the BLAST analysis of the ITS region, the sequences of tub2, tef1-α, and rpb2 were obtained for twelve representative Lasiodiplodia strains for subsequent phylogenetic analysis.

Phylogenetic Analyses
For the phylogenetic identification of Lasiodiplodia species, the combined datasets of four loci, ITS, tub2, tef1-α, and rpb2, comprising 81 Lasiodiplodia isolates, including the sequences of the 12 strains from this study, were analyzed alongside 69 sequences of 36 taxa with their extype specimens.Diplodia seriata (CBS 112555) was included and used as an outgroup taxon.The GTR+G+I model was selected for the concatenated loci.

Pathogenicity and Virulence on Detached Branches and Plants from Certified Commercial Nursery of Persian Lime
Koch's postulates for the Lasiodiplodia strains obtained from Persian lime tissue with dieback were completely corroborated by inoculating disks of PDA with mycelium on detached branches and certified plants.Thirty days after inoculation, all of the isolates belonging to the five Lasiodiplodia species identified in this study were pathogenic to Persian lime, with different degrees of severity, which was not the case for species belonging to other fungal genera (Figure S2).The wood from detached branches exhibited necrotic lesions that extended from both sides of the point of inoculation (Figure 4).

Pathogenicity and Virulence on Detached Branches and Plants from Certified Commercial Nursery of Persian Lime
Koch's postulates for the Lasiodiplodia strains obtained from Persian lime tissue with dieback were completely corroborated by inoculating disks of PDA with mycelium on detached branches and certified plants.Thirty days after inoculation, all of the isolates belonging to the five Lasiodiplodia species identified in this study were pathogenic to Persian lime, with different degrees of severity, which was not the case for species belonging to other fungal genera (Figure S2).The wood from detached branches exhibited necrotic lesions that extended from both sides of the point of inoculation (Figure 4).On Persian lime plants, the Lasiodiplodia strains induced the formation of gum exudations and necrosis in the tissue upward and downward from the point of inoculation (Figure 5).In both cases, the control plants showed no signs of the disease.Lasiodiplodia strains were consistently recovered from affected branches, while none were isolated from healthy control plants, thus satisfying Koch's postulates.On Persian lime plants, the Lasiodiplodia strains induced the formation of gum exudations and necrosis in the tissue upward and downward from the point of inoculation (Figure 5).In both cases, the control plants showed no signs of the disease.Lasiodiplodia strains were consistently recovered from affected branches, while none were isolated from healthy control plants, thus satisfying Koch's postulates.To determine the virulence, the lesion lengths caused by the most aggressive strain of each Lasiodiplodia species from two independent experiments on certified nursery Persian lime plants were averaged (Figure 6).There were significant differences in internal necrosis length produced by the different Lasiodiplodia species (p < 0.05).The longest mean lesions were produced by L. iraniensis, followed by L. pseudotheobromae and L. lignicola, which were the most virulent species.On the other hand, shorter mean lesions were induced by L. theobromae and L. mexicanensis, which were considered the least virulent species.To determine the virulence, the lesion lengths caused by the most aggressive strain of each Lasiodiplodia species from two independent experiments on certified nursery Persian lime plants were averaged (Figure 6).There were significant differences in internal necrosis length produced by the different Lasiodiplodia species (p < 0.05).The longest mean lesions were produced by L. iraniensis, followed by L. pseudotheobromae and L. lignicola, which were the most virulent species.On the other hand, shorter mean lesions were induced by L. theobromae and L. mexicanensis, which were considered the least virulent species.To determine the virulence, the lesion lengths caused by the most aggressive strain of each Lasiodiplodia species from two independent experiments on certified nursery Persian lime plants were averaged (Figure 6).There were significant differences in internal necrosis length produced by the different Lasiodiplodia species (p < 0.05).The longest mean lesions were produced by L. iraniensis, followed by L. pseudotheobromae and L. lignicola, which were the most virulent species.On the other hand, shorter mean lesions were induced by L. theobromae and L. mexicanensis, which were considered the least virulent species.Iran [23], on C. latifolia in Mexico [28], on C. reticulata in Pakistan [56], and recently on C. sinensis in the USA [13].Therefore, research on this species is consistently growing.
In this work, L. lignicola and L. mexicanensis were the least commonly isolated species from symptomatic Persian lime tissues.L. lignicola was initially discovered as saprobic on the dead wood of an unidentified plant in Thailand, where it was named Auerswaldia lignicola [17].However, phylogenetic studies reclassified it as Lasiodiplodia, forming a basal clade for other species [5], and it was also detected in a human keratitis case in a 32-yearold Indian male carpenter in India, in 2012, after trauma caused by a wooden piece [57].Additionally, it was isolated as an endophytic fungus from the healthy tissue of Aquilaria crassna in Laos, suggesting a cosmopolitan role for L. lignicola [58].Recently, L. lignicola was identified as causing canker and dieback diseases on Vangueria infausta subsp.rotundata and Berchemia discolor in lower eastern Kenya [59].Therefore, this is the first report in the world of L. lignicola being associated with dieback symptoms in citrus species.
In the present study, we report for the first time that L. mexicanensis is a causal agent of canker and dieback in Persian lime.Additionally, we analyzed the current status of L. citricola as a causal agent of dieback in Persian lime in Mexico.Our findings clearly demonstrate that strain UACH262, previously identified as L. citricola [28], is actually L. mexicanensis (Figure 3).In this regard, the existence of hybrids between L. parva and L. citricola was previously hypothesized, previously suggested for Lasiodiplodia sp.LACAM1 obtained from Mangifera indica in Peru [60], therefore suggesting that strain UACH262 could be a hybrid, as it groups as a sister clade to L. citricola with high bootstrap/posterior probability (100/0.98).However, Lasiodiplodia sp.LACAM1 was recently identified as L. mexicanensis, a species closely related to L. parva and L. citricola, differing by a few nucleotides in the ITS, tub2, tef1-α, and rpb2 sequences, discarding the hypothesis of LACAM1 being a hybrid [61].According to Cracraft's phylogenetic species concept, this approach does not use data on reproductive isolation, such as hybridization, for the recognition of species taxa; in addition, biogeographic history is important [62].Taking this principle into account, L. citricola was first isolated from Citrus sp. in Iran in 2010 [23], later from Juglans regia [63] and Prunus dulcis [64] in the USA, from Acacia spp.[65] and Persea americana [66] in Italy, and recently from Eriobotrya japonica, Malus domestica, Vitis vinifera, and Juglans regia in China [16].Therefore, phylogenetic and biogeographic data support L. mexicanensis as a species distinct from L. citricola.
On the other hand, in the state of Morelos, Mexico, L. citricola was described as a causal agent of dieback in C. latifolia [67], but, in that study, only the ITS region (KY271187) was used, presenting 100% (540/540) coverage and identity with the extype of L. mexicanensis and 99.79% (476/477) coverage and identity with the extype of L. citricola, respectively.Currently, for the accurate identification of Lasiodiplodia species, the combination of four loci, ITS, tef1-α, tub2, and rpb2, is necessary for reliable resolution [15].Therefore, it can be concluded that L. citricola has not yet been described as associated with canker and dieback in Persian lime (C.latifolia) at this time.
The results of pathogenicity testing showed that the isolates of L. iraniensis were the most virulent, causing the formation of gum exudates and necrosis in the tissue (Figure 5F).These findings are consistent with those of Bautista-Cruz et al. (2019), where L. iraniensis exhibited the highest virulence along with L. subglobosa [28], and Piattino et al. (2024), where L. iraniensis isolates produced the largest necrotic areas compared to Diaporthe spp.[13].L. pseudotheobromae was the second most virulent in Persian lime (Figure 5E), agreeing with results reported by Bautista-Cruz et al. (2019) [22] and Xiao et al. (2021) [28], where it was one of the most aggressive species on citrus shoots.L. lignicola was the third most aggressive species (Figure 5C), with lesion lengths of 23 ± 3.8 mm (Figure 6).This species has

Figure 3 .
Figure 3. Phylogenetic tree of Lasiodiplodia generated from ML analysis of the combined dataset of ITS, tef1-α, tub2, and rpb2.Bootstrap support values for ML ≥ 60% and Bayesian posterior probabilities (PPs) ≥ 0.90 are indicated above at the nodes.Ex−type strains are indicated in bold, and the species are delimited with colored blocks.The isolates collected in the present study are indicated in bold red letters with the nomenclature IXBLT followed by its strain number.The tree was rooted to Diplodia seriata (CBS 112555).

Figure 3 .
Figure 3. Phylogenetic tree of Lasiodiplodia generated from ML analysis of the combined dataset of ITS, tef1-α, tub2, and rpb2.Bootstrap support values for ML ≥ 60% and Bayesian posterior probabilities (PPs) ≥ 0.90 are indicated above at the nodes.Ex−type strains are indicated in bold, and the species are delimited with colored blocks.The isolates collected in the present study are indicated in bold red letters with the nomenclature IXBLT followed by its strain number.The tree was rooted to Diplodia seriata (CBS 112555).

Figure 6 .
Figure 6.Virulence of five Lasiodiplodia species associated with dieback of Persian lime as measured by mean internal lesion lengths (millimeters).Data are lesion sizes measured 30 days after inoculation with mycelium-colonized agar plugs inserted into wounded stem of 18-month-old Persian lime

Figure 6 .Figure 6 .
Figure 6.Virulence of five Lasiodiplodia species associated with dieback of Persian lime as measured by mean internal lesion lengths (millimeters).Data are lesion sizes measured 30 days after inoculation with mycelium-colonized agar plugs inserted into wounded stem of 18-month-old Persian lime Figure 6.Virulence of five Lasiodiplodia species associated with dieback of Persian lime as measured by mean internal lesion lengths (millimeters).Data are lesion sizes measured 30 days after inoculation with mycelium-colonized agar plugs inserted into wounded stem of 18-month-old Persian lime plants from certified nursery.Bars above columns are the standard errors of the means.Columns with the same letter do not differ significantly according to MSD test (p ≤ 0.05).
• C in the dark.The isolates used in this study were stored at −80 • C in 15% glycerol and deposited in the Culture Collection of Phytopathogenic Fungi of the Phytosanitary Diagnosis Laboratory of the Ixtacuaco Experimental Field of the National Institute of Forestry, Agricultural, and Livestock Research (INIFAP), where they are available upon request (https://www.gob.mx/inifap,accessed on 1 July 2024).

Table 1 .
Sequences of primers used in the identification of Lasiodiplodia strains.

Table 2 .
Culture accession numbers, host, location, and GenBank accession numbers of Lasiodiplodia isolates used in the phylogenetic analysis.
T Extype strains; N/A: sequences not available.Newly generated sequences in this study are in bold.BOT: A. M. Ismail, Plant Pathology Research Institute, Egypt.CBS: Centraalbueau voor Schimmelcultures, Utrecht, The Netherlands.CFCC: China Forestry Culture Collection Center, Beijing, China.CGMCC: China General Microbiological Culture Collection Center.CMM: Culture Collection of Phytopathogenic Fungi 'Prof.Maria Menezes' (CMM) at the Universidade Federal Rural de Pernambuco, Brazil.CMW: Tree Pathology Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa.GZCC: Guizhou Academy of Agricultural Sciences Culture Collection, Guizhou, China.IXBLT: Ixtacuaco Experimental Field Fungal Culture Collection of the INIFAP, Mexico.IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Iran.MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand.STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa.UACH: Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University, Mexico.UCD: University of California, Davis, Plant Pathology Department Culture Collection.WAC: Department of Agriculture, Western Australia Plant Pathogen Collection, Australia.ITS: internal transcribed spacer regions; tef1-α: translation elongation factor 1-alpha gene; tub2: beta-tubulin gene; rpb2: DNA-directed RNA polymerase II second largest subunit.