Internal Transcribed Spacer Data Research Articles

First Report of Diaporthe tectonendophytica Causing Leaf Spot on Dalbergia odorifera in Guangxi, China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is a national level II protected plant in China, with extremely high economic value and medical properties (Zhao et al. 2020). In June 2023, an unknown leaf spot was found in a garden land of Pingxiang city, Guangxi, China, and approximately 80% of the plants covered an area of 500 m2 displayed similar symptoms. The spots were grey to white, 4~6 mm in diameter (n=30) with black pycnida on the spots surface (Fig S1, A-D). Multiple disease spots were observed on a single leaf. The pycnida on the lesion were picked and mashed, to make a conidia suspension using sterile water. The conidial solution was then spread onto a potato dextrose agar (PDA) plate containing streptomycin, with 10 mg of streptomycin per 100 mL, and incubated for 3 days at 28°C with a 12 hour photoperiod. Three isolates (GXPX01, GXPX02 and GXPX03) were obtained by re-culturing the colonies on fresh PDA plates. The colony on PDA were white with aerial mycelia (Fig S1, E-F). Black conidiomata developed at 28°C with a 12 hour photoperiod in 20 days (Fig S1, G-H). Alpha conidia were 4.2~6.4 μm × 1.8~2.6 μm (average =5.1 × 2.3 μm, n = 30), mostly bi-guttulate, hyaline, ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth (Fig S1, I). Beta conidia were 15.1~33.5 μm × 1~1.8 μm (average = 24.5 × 1.5 μm, n = 30), filiform, hyaline, curved or hamate, aseptate, base subtruncate (Fig S1, J). Morphological characteristics of the three isolates matched those of Diaporthe spp.(Gomes et al. 2013). The rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1), the calmodulin (CAL), the histone H3 (HIS) and the β-tubulin (TUB2) genes of the three isolates were amplified using the primer pairs ITS4/ITS5, EF1-728F/EF1-986R, CAL-228F/CAL2Rd, CYLH3F/H3-1B, and T1 /CYLTUB1R, respectively (Crous et al. 2004, Sun et al. 2021). The sequences were all deposited in GenBank (accession numbers OR437511 to OR437513 for ITS, OR454965 to OR454967 for TEF1, OR454968 to OR454970 for CAL, OR454971 to OR454973 for TUB2, OR454974 to OR454976 for H3). Sequences had 98.36% to 100% homology with the corresponding sequences of known Diaporthe tectonendophytica strains MFLUCC 13-0471 in the NCBI database. Phylogenetic analysis was based on combined ITS, TEF1, TUB2 and CAL sequences data using MEGA 11 software to construct phylogenetic tree with Maximum Likelihood (Doilom et al. 2017). In the phylogenetic tree, the combined sequences attributed the three isolates to the D. tectonendophytica (Fig S2). The pathogenicity was tested on leaves of 1.5-year-old D. odorifera seedlings. Three leaves were wounded with a sterile needle and individually inoculated with a 5 mm mycelial disk of PDA culture from each isolate. Sterile PDA disks inoculated leaves as a control. The test was repeated three times. The inoculated plants were placed in a greenhouse at 25℃ and 90% humidity, with a photoperiod of 12 hours. Five days after inoculation, necrotic lesions appeared on inoculated leaves and symptoms from all three isolates were the same as those form natural infections ( Fig S1, K-N), whereas all the control remained symptomless (Fig S1, P). The pathogen was reisolated from the inoculated leaves and again identified as D. tectonendophytica, with the same methodology used for the initial identification. D. tectonendophytica was reported to cause plant diseases, such as stem gray blight of red-fleshed dragon fruit (Hylocereus polyrhizus) (Rahim et al. 2021), leaf spots disease on Elaeagnus conferta and Pometia pinnata (Sun et al. 2021). To our knowledge, this is the first report of D. ctonendophytica causing leaf spot disease on D. odorifera.

First Report of Postharvest Fruit Rot Caused by Neofusicoccum parvum on Prunus salicina (Nai Plum) in China.

Nai plum (Prunus salicina var. cordata cv. Younai) is one of the most popular fruit crop in South China. In July 2023, a fruit rot of nai plum with about 5 % disease incidence was observed in a fruit market of Changsha city, Hunan Province, China. Initially, small, brown lesions appeared randomly on the fruit surface, with disease progression, the lesions gradually expanded and developed into soft rot. To isolate possible fungi from rotten fruits, small pieces (2 × 2 mm) from the periphery of 10 infected fruits were surface-sterilized using 70% ethanol for 10 s, rinsed three times in sterile distilled water, air dried, and then placed onto potato dextrose agar (PDA) plates and incubated at 28℃ for three days. Emerging colonies were subcultured by hyphal tiptransfer on fresh PDA. A total of ten isolates with similar morphology were obtained. Fungal colonies were initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidia were hyaline, single celled, ellipsoidal to fusiform, and range from 12.7 to 20.0 μm long (avg. 16.9 ± 2.39 μm) × 5.3 to 7.3 μm wide (avg. 6.3 ± 0.82 μm). These morphological characteristics of these isolates matched those of Neofusicoccum parvum (Phillips et al. 2013). To future confirmation of the identify, the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-a), and beta-tubulin TUB2) genes of two representative isolates (JXNP1 and JXNP2) were amplified and sequenced using primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999; Phillips et al. 2013), and BT2A/BT2B (Glass and Donaldson 1995), respectively. The sequences of both isolates were deposited in GenBank for the ITS (accession nos. OR899331 and OR899332), TEF1-a gene (accession nos. OR909890 and OR909891) and TUB2 gene (accession nos. OR909892 and OR909893). BLAST analysis showed 99-100% identity with the ex-type strain of N. parvum (CMW9081) for ITS, TEF1-a and TUB2. A maximum likelihood phylogenetic tree was constructed using IQtree web server based on combined ITS, TEF1-a and TUB2 data set. The phylogenetic tree revealed that two isolates clustered with N. parvum in a clade with 90% bootstrap support. Based on morphological and molecular data analysis, the isolates were identified as N. parvum. To confirm the pathogenicity, five healthy nai plum fruits were wounded by using a sterile needle after surface sterilization with 75% ethanol, then a 5-mm-diameter mycelial disc of isolate JXNP1 was taped to the wound, the control fruits were taped with sterile agar plugs. All fruits were incubated at 25 ℃ with 80% humidity. After five days, typical naturally occurring fruit rot symptoms appeared on the fruits which inoculated with N. parvum, whereas control fruits remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and comfirmed as N. parvum by morphological and molecular analysis. Previous studies reported that N. parvum caused fruit rot on various common fruits in China, including loquat, kiwifruit and citrus (Lei et al. 2013; Zhai et al. 2019; Zhou et al. 2013). To our knowledge, this is the first report of N. parvum causing postharvest fruit rot on nai plum in China. This finding provides critical insights for the management of the high-risk disease on plum in China.

First Report of Neofusicoccum ribis Causing Cankers and Dieback Diseases on Apricot Trees in Canada and Worldwide.

Apricot trees (Prunus armeniaca L.) with cankers, gummosi and dieback symptoms were observed in a commercial orchard in Niagara-on-the-Lake, Ontario, Canada. In October 2018, up to 44.9% disease incidence (n = 318) was observed on 2-year-old 'Harostar™' trees grafted onto 'Haggith' rootstocks. Fungal colonies were consistently isolated and purified from small sections of wood collected from canker margins of symptomatic trunk and shoot tissue, as described by Ilyukhin et al. (2023). Purified mycelial isolates sharing similar morphological characteristics were categorized into five distinct morphotypes. One representative isolate from each morphotype was used to inoculate excised apricot shoots as described by Ilyukhin and Ellouze (2023). One morphotype displayed necrotic lesions on the shoots consistently yielded abundant white aerial mycelium that turned grey-brown on PDA after 7 days (Figure S1) and produced black pycnidia three weeks following incubtion at 22°C in the dark. Conidia were hyaline, one-celled, fusiform, with dimensions of 19.7 - 24.2 × 3.6 - 4.8 μm (average 22.1 × 4.3 μm, n = 30), the typical morphology of a Neofusicoccum sp. (Crous et al. 2006). Species identification was verified by extracting genomic DNA of the representative isolate M1-105, amplifying and sequencing the internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB2) gene regions with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b. Nucleotide sequences (GenBank Accession No. ITS: OK287034; EF1-α: OK346636; TUB2: OK346633) have 100%, 99.61% and 99.55% identity with Neofusicoccum ribis isolates from different hosts and countries (MT587514, DQ235142, OL455952, respectively). Randomized accelerated maximum likelihood analysis (Stamatakis et al. 2008), using ITS, EF1-α and TUB2 sequence data, clustered M1-105 with the highest bootstrap support values with the N. ribis ex-epitype CBS 115475 (Figure S2). A living culture of M1-105 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252247). Pathogenicity was verified using 5 potted healthy 1-year-old 'Haroblush™' apricot cultivar grafted onto 'Krymsk® 86' rootstocks. Trunks and shoots were inoculated in a shallow wound made by a scalpel with mycelial plugs from a 5-day-old culture of M1-105. Five control trees were inoculated with sterile plugs. Trees were put in an open-air area and watered as needed. Canker symptoms appeared 7 days after inoculation, and spread above and below the inoculation point. Fifteen days post-inoculation, the upper portion of inoculated shoots showed necrosis, gummosis and wilt (Fig. S1). Neofusicoccum ribis was re-isolated from all infected trees and species identity was confirmed by sequencing as described above. Controls remained symptom-free and no fungi were isolated from the wood. Therefore, Koch's postulates were completed. Neofusicoccum ribis was reported to cause branch dieback of olive trees in Spain (Romero et al. 2005) and pistachio in Italy (Corazza et al. 1986), stem blight and dieback of blueberry in Michigan (Heger et al. 2023) and Florida (Wright and Harmon 2010) and postharvest decay of apple fruit from cold storage in Pennsylvania (Jurick et al. 2013). To the best of our knowledge, this is the first report of N. ribis causing canker and shoot dieback of apricot trees in Canada and worldwide. This report reveals N. ribis as a potential threat, causing canker and dieback. Without proper management, it could lead to significant losses in apricot orchards and the stone fruit industry. This study paves the way for crucial research on N. ribis outbreaks and effective disease control.

Xenoacremonium palmarum sp. nov., a novel species associated with Phoenix dactylifera in Iran

The date palm (Phoenix dactylifera L.) is one of the most important fruit crops in Iran, where it has been cultivated in the south, southeast, and southwest of Iran. Root fragments of date palm showing root rot symptoms were collected from different areas in Khuzestan and Bushehr provinces to isolate their fungal associations. Accordingly, six isolates of a new species, Xenoacremonium palmarum, were obtained, herein described and illustrated. This new species is identified based on both morphology and phylogenetic analyses of combined ITS (the internal transcribed spacer region and intervening 5.8S nrRNA), tub2 (β-tubulin) and tef1α (translation elongation factor 1-alpha) sequence data. Our isolates grouped in a highly-supported clade, distinct from previously known species of Xenoacremonium. The new species and X. allantoideum differ from both X. falcatum and X. minutisporum in not having lateral phialidic pegs on their somatic hyphae. In addition, conidia in X. falcatum are more curved than those in X. allantoideum, X. palmarum and X. minutisporum. New species, X. palmarum, differs from X. allantoideum in having longer conidia and lacking mycelial ropes.

Mycena cinereoalba sp. nov. from Qinghai province, China

From specimens collected in Sanjiangyuan (Source of Three Rivers) region, Qinghai Province, China, Mycena cinereoalba is described based on its morphological characteristics and a molecular phylogenetic analysis using the internal transcribed spacer (ITS) region and nuclear ribosomal RNA large subunit (nrLSU) sequences. The phylogenetic relationship inferred from the ITS and nrLSU sequence data matrices confirmed that this new species is different from other species of Mycena for which DNA sequences are available. The species are comprehensively described and illustrated in detail with photographs. Phylogenetic relationships among the species were analyzed, and two phylograms were provided in this study.

First Report of Anthracnose Caused by Collectotrichum higginsianum on Rumex crispus in China.

Rumex crispus L. is a perennial herb with medicinal properties derived from its roots and whole plant (Bhandari et al. 2022). In December 2022, symptoms of anthracnose were observed in approximately 40% of naturally occurring R. crispus plants in Longquan Reservior, Nanchang city (115°53' N, 28°43' E), Jiangxi Province, China. Initially, red lesions appeared randomly on various parts of the leaf blade, which gradually became dry and brown at the center, eventually leading to leaf death. To isolate the fungal pathogen responsible, ten symptomatic leaves were randomly collected and their lesions were cut into small pieces (4 × 4 mm). The leaf fragments were surface-sterilized in 70% ethanol for 45 s and then in 1% NaClO for 45 s. The leaf pieces were rinsed three times with sterile distilled water. The surface-sterilized leaf pieces were then placed onto potato dextrose agar (PDA) and incubated at 28 ℃, dark condition for 3 days. Twelve isolates were obtained, characterized by a milky white and uneven growth pattern with a white root-like structure branching out at the edge, along with scattered black deposits on the bottom of the plate. Conidiogenous cells cylindrical, smooth-walled, hyaline, 9.3-23.2 × 3.6-4.2 μm. Conidia elliptical, aseptate, smooth-walled, with one end blunt and the other truncate, ranging in size from 10.4 to 22.3 (mean 16.7) μm in length and 3.2 to 5.0 (mean 4.1) μm in width (n = 50), which are consistent with the characteristics of the members of Colletotrichum destructivum species complex (Damm et al. 2014). To accurately identify the strain, three representative isolates, namely JFRL 03-930, JFRL 03-931, and JFRL 03-935, were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GADPH), histone3 (HIS3), and beta-tubulin (TUB2) genes were amplified and sequenced using specific primer pairs, including ITS5/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, GYLH3F/CYLH3R, and T1/Bt2b (Damm et al. 2014). All sequences were deposited in GenBank with accession numbers OQ560476-OQ560478 (ITS), OQ576154-OQ576156 (ACT), OQ576157-OQ576159 (CHS), OQ576160-OQ576162 (HIS3), OQ576163-OQ576165 (GADPH), and OQ576166-OQ576168 (TUB2). A maximum likelihood phylogenetic tree was constructed using IQtree v1.5.6 based on the combined ITS, ACT, CHS, GAPDH, HIS3 and TUB2 data set (Nguyen et al. 2015), The phylogenetic tree showed that the three isolates clustered with C. higginsianum in a clade with 91% bootstrap support. Based on morphology and molecular characters, the isolates were identified as C. higginsianum of the C. destructivum species complex. To confirm the pathogenicity, One-year-old R. crispus were collected from the wild and potted in an climate chamber. Six healthy leaves of R. crispus were surface sterilized with 70% ethanol and wounded by sterile needle, and a 20-μl conidial suspension (3×105 conidia/ml) of the isolate JFRL 03-931 was inoculated on the wound. Another set of six leaves of R. crispus was inoculated with distilled water as controls. The potted plants were incubated under conditions of 25 ℃ and 80% humidity. After 10 days, reddish brown spots were observed on all inoculated leaves, while the control leaves remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated leaves and confirmed as C. higginsianum by morphological and molecular analysis. It has been reported that C. higginsianum caused anthracnose disease on several cruciferous vegetables, Boehmeria nivea and Rumex acetosa in China (Damm et al. 2014; Wang et al. 2011; Patel et al. 2014; Zhang et al. 2018). But to our knowledge, this is the first report of C. higginsianum casued anthracnose on Rumex crispus in China. Therefore, we should pay more attention to this pathogen and develop appropriate control strategies.

Occurrence of Colletotrichum truncatum Causing Foliar Spot on Sesame (Sesamum indicum) in Mexico.

Sesame (Sesamum indicum L.) is an oilseed crop that present agronomic advantages and nutritional contributions in regions where water and soil fertility are limiting. In September 2020 and October 2022, anthracnose symptoms were observed on sesame fields in Mocorito (25°29'04"N;107°55'03"W) and Guasave (25°45'40"N;108°48'44"W), Sinaloa, Mexico. The disease incidence was estimated at up to 35 % (10 has) in five fields. Twenty samples were collected with symptoms on the leaves. On leaves, lesions were irregular and necrotic. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as a representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Product Development Center at the National Polytechnic Institute under the accession number IPN 13.0101. On PDA, colonies were flat with an entire margin, initially white, then dark gray with black acervuli and setae. The growth rate was 9.3 mm/day. Conidia (n=100) on PDA were hyaloamerosporae, 17.5- 22.7 × 3.6-4.5 μm, smooth-walled, falcated and pointed at both ends, with granular content. Acervuli showed setae acicular (2-3 septate setae) tapered to the apex. The mycelial appressoria were brown, obclavate and irregular. Morphological features matched those of the Colletotrichum truncatum species complex (Damm et al. 2009). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR (Weir et al. 2012) and sequenced. The sequences were deposited in GenBank under accession nos. OQ214919 (ITS), OQ230773 (ACT), and OQ230774 (GAPDH). BLASTn searches in GenBank showed 100%, 100%, and 100% identity to MN842788 (ITS), MG198003 (ACT), and MF682518 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on the Maximum Likelihood method and Bayesian Inference including published ITS, ACT, and GAPDH sequence data for C. truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate IPN 13.0101 was placed in the same clade of C. truncatum. Pathogenicity of the isolate IPN 13.0101 was verified on 15 sesame seedlings leaves (Dormilon variety) (15-day-old) disinfected with sodium hypochlorite and sterile water. Each leave was inoculated with 200 µL of a conidial suspension (1 × 106 spores/mL). Five plants non inoculated served as controls. All plants were kept in a moist chamber for two days, and subsequently transferred to a shade house where the temperature ranged from 25 to 30°C. All inoculated leaves developed irregular and necrotic lesions ten days after inoculation, whereas no symptoms were observed on the control leaves. The fungus was consistently re-isolated from the diseased leaves, fulfilling Koch´s postulates. The experiment was conducted twice with similar results. Colletotrichum spp. has been previously reported (Farr and Rossman, 2023) to cause sesame anthracnose in Mexico (Alvarez, 1976), Thailand (Giatgong, 1980) and Cuba (Arnold, 1986), but this is the first report of C. truncatum causing sesame anthracnose in Mexico. This disease is a recurrent problem in sesame fields in Sinaloa, therefore further studies are required to understand its impact.

First Report of Pestalotiopsis trachicarpicola Causing Leaf Blight on Panax notoginseng in China.

Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K.M. Feng is a Chinese herbal medicinal plant for treating diseases of the central nervous system and cardiovascular system, widely used as a medicine and health-care product. In May 2022, leaf blight disease was found on leaves of 1-year-old P. notoginseng in the plantings （27.904°N, 112.918°E） of Xiangtan City (Hunan) with an area of 104 m2. Over 400 plants were investigated, up to 25% of the plants were symptomatic. From the margin of the leaf, the initial symptoms of water-soaked chlorosis and following dry yellow with slight shrinkage appeared. Later, leaf shrinkage became serious and chlorosis enlarged gradually, leading to leaf death and abscission. To identify the causal agent, 20 leaf lesions (4 mm2) collected from 20 individual 1-year-old plants were sterilized with 75% ethanol for 10 s, 5% NaOCl for 10 s, rinsed in sterilized water three times, placed on potato dextrose agar (PDA) with lactic acid (0.125%) for inhibition the growth of bacteria, and incubated at 28°C for 7 days (Fang, 1998). Five isolates were obtained from 20 leaf lesions of different plants with the isolation rate of 25% and purified by single sporing, which have similar colony and conidia morphology characteristics. One isolate PB2-a was selected randomly for further identification. Colonies of PB2-a on PDA were white with cottony mycelium, developing concentric circles (top view) or light yellow (back view). Conidia (23.1 ± 2.1 × 5.7 ± 0.8 µm, n=30)were fusiform, straight or slightly curved and contained conic basal cell, three light brown median cells and hyaline conic apical cell with appendages. The rDNA internal transcribed spacer (ITS), the translation elongation factor 1-alpha (tef1), and the β-tubulin (TUB2) genes were amplified from genomic DNA of PB2-a using primers ITS4/ITS5 (White et al. 1990), EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), and Bt2a/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997), respectively. BLAST search of sequenced ITS (OP615100), tef1 (OP681464) and TUB2 (OP681465) exhibited > 99% identity with the type strain of Pestalotiopsis trachicarpicola OP068 (JQ845947, JQ845946 and JQ845945). Phylogenetic tree of the concatenated sequences was constructed based on the maximum-likelihood method using MEGA-X. Isolate PB2-a was identified as P. trachicarpicola based on morphological and molecular data (Maharachchikumbura et al. 2011; Qi et al. 2022). PB2-a was tested for pathogenicity three times to confirm Koch's postulates. Twenty healthy leaves on 20 1-year-old plants were punctured with sterile needles and inoculated with 50 µl of conidial suspension (1×106 conidia/ml). The controls were inoculated with sterile water. All plants were placed in a greenhouse at 25°C under 80% relative humidity. After 7 days, all inoculated leaves developed leaf blight symptoms identical to those described above, whereas the control plants kept healthy. P. trachicarpicola were reisolated from infected leaves, and identical to those of the originals based on the colony characteristics and the sequenced data of ITS, tef1 and TUB2. P. trachicarpicola was reported as a pathogen of leaf blight on Photinia fraseri (Xu et al. 2022). To our knowledge, this is the first report of P. trachicarpicola causing leaf blight on P. notoginseng in Hunan, China. Leaf blight is one of the destructive diseases in P. notoginseng production, identification of the pathogen will be useful to develop effective disease management and protect P. notoginseng, a medical plant with economic value.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Fruit Rot on Indian Jujube in China.

Indian jujube (Ziziphus mauritiana Lamarck), is one of the most popular fruit crops in South China. In March 2023, a fruit rot of indian jujube with about 5% disease incidence was observed in two supermarkets of Nanchang City, Jiangxi Province, China. Initially, the symptoms appeared as slightly brown spots on the fruit surface, with disease progression, the lesions gradually expanded and covered with a layer of hyphae. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected using 1% sodium hypochlorite for 30 s, rinsed three times in sterilized distilled water, air dried, and then aseptically placed onto potato dextrose agar (PDA) media and incubated at 25°C for three days. A total of ten single spore isolates with similar morphology were obtained. Colonies of these consisted of initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidiogenous cells were smooth, hyaline, cylinder-shaped, and holoblastic. Conidia were ellipsoidal, top and base-rounded, and thick-walled, immature conidia were colorless, hyaline, and aseptate, compared with dark brown color of the mature conidia, which were one-septate with longitudinal striations, ranging in size from 22.8 to 31.8 (mean 27.6) µm in length and 12.2 to 20 (mean 14.6) µm in width. The morphological characteristics were consistent with the characteristics of the Lasiodiplodia species (Phillips et al. 2013). To accurately identify the strain, three representative isolates, namely JFRL 03-1147, JFRL 03-1148, and JFRL 03-1149, were selected for further identification. The internal transcribed spacers (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB2) genes/regions were amplified and sequenced using primers ITS1/ITS4, EF1-688F/EF1-1251R, and Bt2a/Bt2b, respectively (Chen et al. 2021). These nucleotide sequences were deposited in GenBank with accession numbers OQ804425-OQ804427 (ITS), OQ818097-OQ818099 (TEF1-α), and OQ818100-OQ818102 (TUB2). A BLASTn homology search for these nucleotides showed 99-100% identity to ITS (EF622077, 487 nt/487 nt), TEF1-α (EF622057, 306 nt/307 nt) and TUB2 (EU673111, 434 nt/434 nt) sequences of Lasiodiplodia pseudotheobromae CBS 116459 (ex-type). The maximum likelihood analyses were performed for the combined ITS, TEF1-α and TUB2 data set using IQtree web server (Trifinopoulos et al. 2016). The phylogenetic tree showed that the three isolates clustered with Lasiodiplodia pseudotheobromae in a clade with 99% bootstrap support. Therefore, the fungus was identified as L. pseudotheobromae based on morphological and molecular characteristics. To evaluate pathogenicity, 4 healthy fruits of indian jujube were surface sterilized with 75% ethanol and wounded by sterile needle, and a 5-mm-diameter agar with 5-days-old mycelium of the isolate JFRL 03-1148 cultured on PDA at 25°C was put on the wound. Another set of 4 fruits was inoculated with sterile agar plugs as controls. The fruits were cultured at 25℃ and 85% relative humidity, and the test was repeated twice. These fruits inoculated with L. pseudotheobromae showed similar rot symptoms after 3 days, while the control group remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as L. pseudotheobromae by morphological and molecular analysis. L. pseudotheobromae has previously been reported causing fruit rot on citrus, mango and papaya (Alam et al. 2021; Chen et al. 2021; Netto et al. 2014). But to our knowledge, this is the first report of L. pseudotheobromae caused postharvest fruit rot on indian jujube in China. Therefore, managers should pay more attention to postharvest fruit rot disease caused by L. pseudotheobromae, and formulate appropriate disease control measures to reduce its losses.

First Report of Fusarium citri Causing Postharvest Fruit Rot of Chayote in Mexico.

In November 2018, symptoms of brown rot were observed on chayote (Sechium edule) var. nigrum spinosum with a 20% disease incidence of 120 harvested fruits in the National Germplasm Bank of Sechium edule, located in the Centro Regional Universitario Oriente (CRUO) from the Chapingo Autonomous University (Huatusco, Veracruz, Mexico). For fungal isolation, pieces from symptomatic fruits were surface disinfected by immersion in a 1.5% NaClO solution for 2 min, rinsed in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA) amended with kanamycin sulfate, and incubated at 25ºC. Fusarium-like colonies were consistently isolated on PDA and five monoconidial isolates were obtained. A representative isolate was selected for morphological characterization, phylogenetic analysis, and pathogenicity tests. On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pale brown pigment in the center at 7 days of incubation at 25℃ in darkness. Macroconidia (n= 100) were hyaline, falcate, with 4 to 5 septa, measuring 23.9 to 31.9 × 2.9 to 4.2 μm, and foot-shaped basal cells. Microconidia and chlamydospores were absent. Morphological features were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). The isolate was deposited as FUS2 in the Culture Collection of Phytopathogenic Fungi of the Laboratory of Plant Pathology at the Colegio de Postgraduados. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, partial sequences of translation elongation factor 1-alpha (EF1-α), and the second-largest subunit of RNA polymerase II (rpb2) genes were amplified, and sequenced with the primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. DNA sequences were edited in BioEdit 7.2 and compared with those in the NCBI nucleotide database. Alignments were implemented in MEGA X using reference sequences from Fusarium spp. A phylogenetic tree, including published ITS, EF1-α, and rpb2 sequence data, was constructed for the Fusarium incarnatum-equiseti species complex (FIESC) based on Maximum Likelihood. The sequences were deposited in GenBank (accession nos. ON878083, ON890421, and ON890420). The phylogenetic analysis grouped the isolate FUS2 within the F. citri clade. Pathogenicity of the fungus was verified on 10 healthy chayote fruits var. nigrum spinosum previously disinfested by immersion in a 1% NaClO solution for 3 min and washed in sterile water. A total of 5 mL of a conidial suspension (1 × 106 spores/ml) was sprayed on each whole fruit. Ten control fruit were sprayed with sterile distilled water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 30 days. All inoculated fruits developed water-soaked brown lesions (3 to 5 cm in diameter) covered with white mycelium at 15 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Fusarium citri has been associated with Capsicum sp. and mandarin orange in China, Triticum sp. in Iran, alfalfa in Denmark, and lettuce in the Czech Republic and Italy (Farr and Rossman 2022). To our knowledge, this is the first report of F. citri causing postharvest fruit rot of chayote in Mexico and worldwide.

First Report of Phytophthora infestans Causing Late Blight on Pepino (Solanum muricatum) in China.

Solanum muricatum, known as pepino or melon pear, is a species of evergreen shrub grown for its sweet edible fruits, which was introduced in Yunnan about 20 years ago. Since 2019 to now, serious blight disease was observed on foliages, haulms and fruits of pepino plants in Shilin (25°N, 103°E), which is the biggest pepino production area in China. The symptomatic blighted plants showed water-soaked and brown foliar lesions, haulm brown necrosis, black-brown and rotting fruits, and overall plant decline. The samples with typical disease symptoms were collected for pathogen isolation. After surface sterilization, disease samples were cut into small pieces and placed on rye sucrose agar medium amended with both 25 mg/liter rifampin and 50 mg/liter ampicillin, then incubated in the dark at 25℃ for 3 to 5 days. White fluffy colonies of mycelia that grew from the edge of diseased tissues were further purified and subcultured on rye agar plates. All purified isolates were identified as Phytophthora spp. based on morphological characteristics (Fry 2008). Sporangiophores were nodular and sympodial branches with swellings at the points where sporangia were attached. Sporangia that were hyaline and the average size were 22×40 μm were formed on the tip of sporangiophores and appeared as subspherical, ovoid, ellipsoid or lemon shaped with half-papillate on the spire. Mature sporangia were easilly detached from sporangiophores. For pathogenicity tests, healthy leaves, haulms and fruits of pepino were inoculated with 1×104 cfu/ml zoospore suspension of Phytophthora islolate (RSG2101), and controls were treated with sterile distilled water, respectively. After 5 to 7 days postinoculation, all Phytophthora-inoculated leaves and haulms showed water-soaked and brown lesions with white mold layer, fruits showed dark-brown firm lesions which got expanded and rotted the entire fruit. The symptoms were same as those occurred in natural fields. In contrast, no disease symptoms appeared in the control tissues. Phytophthora isolates could be reisolated and showed same morphological characteristics from the infected tissues of leaves, haulms, and fruits, sufficing Koch's postulates. Two common molecular targets of the internal transcribed spacer (ITS) region of ribosomal DNA and partial cytochrome c oxidase subunit II (CoxII) of the Phytophthora isolate (RSG2101) were amplified and sequenced with primers ITS1/ITS4 and FM75F/FM78R (Kroon et al. 2004). The ITS and CoxII sequence data were deposited in GenBank under accession numbers OM671258 and OM687527, respectively. Blastn analysis of both ITS and CoxII sequences showed 100% identity with isolates of P. infestans (MG865512, MG845685, AY770731 and DQ365743, respectively). Phylogenetic analysis also indicated that RSG2101 isolate and known P. infestans isolates localized in the same evolutionary branch based on sequences of ITS and CoxII, respectively. Based on these results, the pathogen was identified as P. infestans. It is known that P. infestans infection of pepino occurrs in Latin America and then it was recorded in other parts of the world such as New Zeeland and India (Hill, 1982; Abad and Abad,1997; Mohan et al. 2000).To our knowledge, this is the first report of late blight on pepino caused by P. infestans in China, which will be helpful to develop efficient blight management strategies on pepino.

First Report of Leaf Blight Caused by Colletotrichum karsti on Peppermint (Mentha x piperitavar. citrata) in Mexico.

Spearmint (Mentha x piperita var. citrata (Ehrh.) Briq.: Lamiaceae) is an aromatic herb widely cultivated owing to its industrial properties. In June 2020, symptoms of leaf blight were observed on 1,500 peppermint plants in a commercial nursery located in Cuautla (18°52'18"N 98°57'58"W), Morelos, Mexico. The incidence of the disease was 89%. Symptoms were initially observed as irregular, small black necrotic spots, that grew rapidly until the leaves were blighted. Fungal isolation was done using diseased leaf tissue on potato dextrose agar (PDA) as described by Ayvar-Serna et al. (2020) and Colletotrichum-like colonies were obtained. Six isolates were purified by single spore culture and only a single morphotype was obtained. One isolate was used for pathogenicity tests, morphological characterization, and multilocus phylogenetic analysis. The isolate (accession no. UACH449) was deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University. Colonies in PDA grow at a rate of 7.0-10.0 mm/d. After 14 days, the colony was white to orange, and conidia (n =100) were hyaline, cylindrical, and straight with rounded ends, measuring 15.0-17.0 × 4.5-6.5 μm. Appressoria were brown and bullet-shaped. In 28-day-old colonies, the formation of perithecia was observed. Asci were hyaline, unitunicate, 8-spored, fasciculate, and cylindrical to clavate. Ascospores (n =100) were hyaline, unicellular, allantoid, inaequilateral, often straight on the inner side, apices rounded, arranged biseriately within the asci, and measured 14-19 × 4.0-7.5 μm. Morphological features of the isolate placed it tentatively within the Colletotrichm boninense species complex (Damm et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) (Damm et al. 2012) genes were amplified and sequenced. A phylogenetic tree including published ITS, CAL, GAPDH, and ACT data for Colletotrichum species was constructed and the isolate UACH449 was grouped in the clade of Colletotrichum karsti. Sequences were deposited in GenBank under the accession numbers: ITS, OL825605; CAL, OL855890; GAPDH, OL855891 and ACT, OL855889. Pathogenicity was tested by spraying a suspension of 1 × 10^5 conidia/ml, onto eight healthy peppermint plants 30-days-old var. citrata, while eight control plants were sprayed using sterile distilled water. All plants were kept at 25 +/- 2°C and 70% RH. The characteristic symptoms of the disease were observed seven days after inoculation, while control plants remained symptomless. The pathogenicity test was repeated twice. The fungus was consistently reisolated from the eight inoculated plants and was morphologically identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. To date, this pathogen has not been reported on peppermint (Farr and Rossman, 2022). To our knowledge, this is the first report of Colletotrichum karsti causing foliar blight on peppermint worldwide. According to our field observations, this disease is a threat to the production of peppermint plants.

Pseudocercospora rhododendricola Causing Leaf dark Spot on Rhododendron pulchrum by the first phylogenetic analyses.

Rhododendron pulchrum Sweet is a famous ornamental flower in China. In December 2020, a leaf spot disease was observed on cv. Maojuan in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The spots were irregular and distributed on both sides of the main vein. They were dark to black, and their borders were obvious. The coalescence of the spots eventually led to leaf wilt. The disease incidence was 100% (n = 100, about 50 ha ). Thirty infected leaves were collected from the field, and the margin of the diseased tissues was cut into 2 mm × 2 mm pieces. Samples were surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively. They were rinsed thrice with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 ℃. After 5 days, fungal colonies appeared on the PDA. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates(RSP-1, RSP-2, and RSP-3) were obtained and the colonies of isolates were preserved in glycerol (15%) at -80 °C deposited at the Museum of Guangdong Ocean University. The morphology of these three isolates was consistent, and their sequences showed 100% homology according to ITS, TEF1, and ACT analysis results. The colonies grew to approximately 5 cm in diameter after 10 days. They showed olive green with off-white aerial mycelia. Stromata and conidia were observed on leaf lesions. Stromata were olivaceous brown. Conidia were solitary, cylindrical to narrowly obclavate, mildly curved, obtuse to rounded at the apex, and 1- to 3-septate; they had dimensions of 20 to 60 × 2.0 to 3.0 μm (n= 30). These morphological characteristics were not different from the description of Pseudocercospora rhododendricola (J.M. Yen) Deighton (Liu et al. 1998). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS4/ITS5, EF1/EF2, and ACT-512F/ACT-783R, respectively (White et al., 1990; O'Donnell et al. 1997). The sequences of the isolateRSP-1 were deposited in the GenBank (ITS, MW629798; TEF1, MW654168; and ACT, MW654170). BLAST analysis showed that the sequences of P. rhododendricola were submitted to GenBank for the first time by the author of this paper. A phylogenetic tree was generated based on the concatenated data of ITS, TEF1, and ACT sequences from GenBank by the Maximum Likelihood method. The isolates were closest to Pseudocercospora sp. CPC 14711 (Crous et al., 2013). Phylogenetic and morphological analyses identified the isolates as P. rhododendricola. Pathogenicity tests were conducted in a greenhouse at 24°C-30℃ with 80% relative humidity. Healthy cv. Maojuan were grown in pots. Unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control) (5 leaflets per plant, 3 plants, 2-month-old plants). The test was performed thrice. Disease symptoms were found on the leaves after 2 weeks, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolatesby morphological and ITS analyses. P. rhododendricola was the cause of leaf spot of Rhododendron sp. from Singapore (Liu et al., 1998). For the first time, this pathogen was identified by combining phylogenetic and morphological analyses. The sequences in this study would be used as the reference sequences for further studies.

First Report of Ceratobasidium sp. (AG-A and AG-G) Causing Root Rot and Stem Canker of Common Bean in Mexico.

Common bean (Phaseolus vulgaris L.), is a grain legume widely cultivated worldwide for its edible dry seeds and pods. In February 2021, root rot symptoms were observed in two common bean (cv. Azufrado Higuera) fields located in Ahome (25º96´19¨N, 109º33´42¨W) and Guasave (25º71´85¨N, 108º78´50¨W) municipalities in Sinaloa, Mexico. Diseased plants showed reduced growth, dark brown canker at the base of the stem, root rot, as well as the absence of secondary roots. The disease incidence was estimated up to 35%. For fungal isolation, symptomatic roots were surface sterilized with 1% sodium hypochlorite for 2 min, rinsed with sterilized distilled water two times, and blotted dry on sterile filter paper. Small fragments of diseased roots were placed on PDA medium and incubated at 25°C in darkness for 3 days. Rhizoctonia-like colonies were consistently obtained and 10 isolates were purified by the hyphal-tip method. Colonies on PDA were white initially and then turned brown after 15 days of incubation. The septate hyphae were 3.9 to 6.3 μm in width and branched at right angles with a septum near the point of branching. Microscopic examination by Safranine-O staining showed two nuclei per cell. The morphological features of the isolates resembled those of Ceratobasidium (Sneh et al. 1991). The two Ceratobasidium isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF395 and FAVF396). For molecular identification, genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region and partial fragments of the second largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with the primer pairs ITS5/ITS4 (White et al. 1990) and RBP2-980F/RPB2-7cR (Liu et al. 1999), respectively. The ITS sequences (accession nos. ON630914 and ON630915) showed 99.66 and 99.01% identity with Ceratobasidium sp. (AG-A) from the USA (OM045887) and Ceratobasidium sp. (AG-G) from China (HM623627), respectively. Whereas, the rpb2 sequences (OM258171 and OM258172) showed 94.10 and 95.74% identity with Ceratobasidium sp. (AG-A) from Serbia (MT1267888) and Ceratobasidium sp. (AG-G) from Japan (DQ301701), respectively. A phylogenetic tree based on Maximum Likelihood and including combined ITS and rpb2 sequences data for Ceratobasidium spp. was generated. The phylogenetic tree grouped the isolates FAVF395 and FAVF396 within the Ceratobasidium sp. AG-A and AG-G clades, respectively. Pathogenicity tests for each isolate were performed by inoculating 10 healthy common bean seedlings (15-day-old) grown in pots. A total of 50 ml of a mycelial suspension adjusted to a concentration of 1 × 105 mycelial fragments/ml were directly placed on the stem base of each plant. Five uninoculated common bean seedlings were used as control. All plants were kept in a greenhouse for 15 days at temperatures ranging from 22 to 32°C. Root rot and stem canker symptoms appeared on inoculated seedlings after 10 days, whereas control plants remained symptomless. Fungi were reisolated from the infected roots and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. Ceratobasidium sp. has been previously reported as the causal agent of root rot of watermelon in Sonora, Mexico (Meza-Moller et al. 2014; Farr and Rossman 2022). To our knowledge, this is the first report of Ceratobasidium sp. causing root rot of common bean in Mexico. Further monitoring should be performed to quantify yield impacts and develop effective management strategies for this disease.

Phylogeographic investigation of the bladder grasshopper Bullacris unicolor (Orthoptera Pneumoroidea) in South Africa

There are several factors, such as genetic drift, gene flow and migration that affect the population genetic structure and phylogeographic distribution of genetic lineages within single species. Previous studies of the bladder grasshoppers, Bullacris unicolor of South Africa, showed divergence in mitochondrial CO1 (cytochrome c oxidase subunit 1) haplotype diversity and significant genetic structure. In this study, we revisit these findings adding more samples from different locations and using mitochondrial CO1 and Internal transcribed spacer (ITS) gene sequences. We tested the hypothesis that the western, northern and eastern distribution ranges of B. unicolor show different population genetic patterns, corresponding with isolation-by-distance. Mitochondrial CO1 and ITS data were collected for 99 individuals from 12 localities across the Western, Northern and Eastern sides of South Africa. Overall, significant variation in genetic structure was found across the localities as indicated by F ST analyses. Haplotype and phylogeographical analyses suggested that restricted gene flow played a role in shaping current genetic patterns, although isolation-by-distance was not supported, as confirmed by Mantel tests. Phylogenetic trees of both genetic sequences revealed two major clades, with western and northern sides. Also, the major clades exhibit a few sub-clades within the localities, showing other factors shaping the genetic structure of B. unicolor include the geographical barriers, and most likely due to changes in habitat specificity and habitat fragmentation. Taken together, this study aims to contribute information on the population structure and genetic diversity of B. unicolor populations across South Africa.

Molecular identification of a new species of Rhigonema (Nematoda: Rhigonematidae) and phylogenetic relationships within the infraorder Rhigonematomorpha

BackgroundThe infraorder Rhigonematomorpha comprises a group of obligate parasitic nematodes of millipedes (Arthropoda: Diplopoda). The current species identification of Rhigonematomorpha nematodes remains mainly based on morphological features, with molecular-based identification still in its infancy. Also, current knowledge of the phylogeny of Rhigonematomorpha is far from comprehensive.MethodsThe morphology of Rhigonematomorpha nematodes belonging to the genus Rhigonema, collected from the millipede Spirobolus bungii Brandt (Diplopoda: Spirobolida) in China, was studied in detail using light and scanning electron microscopy. Five different genetic markers, including the nuclear small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S) regions and the mitochondrial cox1 and cox2 genes of these Rhigonematomorpha nematodes collected from China and Rhigonema naylae collected from Japan were sequenced and analyzed using Bayesian inference (BI) and Assemble Species by Automatic Partitioning (ASAP) methods. Phylogenetic analyses that included the most comprehensive taxa sampling of Rhigonematomorpha to date were also performed based on the 18S + 28S genes using maximum likelihood (ML) and BI methods.ResultsThe specimens of Rhigonema collected from S. bungii in China were identified as a new species, Rhigonema sinense n. sp. Striking variability in tail morphology was observed among individuals of R. sinense n. sp. ASAP analyses based on the 28S, ITS, cox1 and cox2 sequences supported the species partition of R. sinense n. sp. and R. naylae, but showed no evidence that the different morphotypes of R. sinense n. sp. represent distinct genetic lineages. BI analyses also indicated that R. sinense n. sp. represents a separated species from R. naylae based on the cox1 and cox2 genes, but showed that R. naylae nested in samples of R. sinense n. sp. based on the ITS and 28S data. Phylogenetic results showed that the representatives of Rhigonematomorpha formed two large clades. The monophyly of the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema was rejected. The representatives of the family Ransomnematidae clustered together with the family Hethidae with strong support.ConclusionsA new species of Rhigonematomorpha, R. sinense n. sp. is described based on morphological and molecular evidence. ASAP analyses using 28S, ITS, cox1 and cox2 data indicate the striking variability in tail morphology of R. sinense n. sp. as intraspecific variation, and also suggest that partial 28S, ITS, cox1 and cox2 markers are effective for molecular identification of Rhigonematomorpha nematodes. The phylogenetic results support the traditional classification of Rhigonematomorpha into the two superfamilies Rhigonematoidea and Ransomnematoidea, and indicate that the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema are non-monophyletic. The present phylogeny strongly supports resurrection of the family Brumptaemiliidae, and also indicates that the family Ransomnematidae is sister to the family Hethidae.Graphical

First Report of Colletotrichum truncatum Causing Anthracnose of Guar (Cyamopsis tetragonoloba) in Mexico.

Guar (Cyamopsis tetragonoloba), is an annual legume belonging to the Fabaceae family and it is grown mainly for industrial purposes and also as an ingredient for animal feed. In September 2021, anthracnose symptoms were observed on guar fields distributed in Guasave, Sinaloa, Mexico. Disease incidence was estimated up to 15%. Diseased plants exhibited symptoms on leaves and pods. On leaves, lesions were irregular, necrotic, and often surrounded by a dark brown halo. On pods, necrotic and sunken lesions were developed. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under the accession number FAVF642. Colony on PDA was flat with an entire margin, dense, initially grayish white, then became dark gray with black microsclerotia and setae. Conidia (n= 50) were curved, hyaline, aseptate, with granular content, and measuring 20.4 to 25.8 × 2.8 to 3.9 μm. Setae were dark brown, straight, and septate. Morphological features matched those of Colletotrichum truncatum (Damm et al. 2009). For morphological identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR (Weir et al. 2012), and sequenced. The resulting sequences were deposited in GenBank under the accession nos. OM616022 (ITS), OM630461 (ACT), and OM630462 (GAPDH). BLASTn searches in GenBank showed 100%, 99.49%, and 99.15% identity to MT583079 (ITS), MG198003 (ACT), and MG703491 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on maximum Likelihood method and including published ITS, ACT, and GAPDH sequence data for Colletotrichum truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate FAVF642 was placed in the same clade of C. truncatum. Pathogenicity of the isolate FAVF642 was verified on 10 guar seedlings (15-day-old) by spraying a conidial suspension (1 × 106 spores/mL) onto leaves until runoff. Five plants noninoculated served as controls. All plants were kept in a moist chamber for 2 days, and subsequently transferred to a shade house where the temperature ranged from 20 to 30°C. The experiment was conducted twice with similar results. All inoculated leaves developed irregular and necrotic lesions 8 days after inoculation, whereas no symptoms were observed on the control leaves. The fungus was consistently re-isolated from the diseased leaves, fulfilling Koch´s postulates. Colletotrichum truncatum has been previously reported to cause guar anthracnose in India (Farr and Rossman 2022). To our knowledge, this is the first report of C. truncatum causing guar anthracnose in Mexico. This disease is an emerging problem in guar fields in Sinaloa, therefore further studies are required to understand its occurrence and impact in Mexico.

Occurrence of the Fusarium incarnatum-equiseti Species Complex Causing Fusarium Head Blight of Wheat in Mexico.

Fusarium head blight (FHB) is one of the most important diseases affecting wheat production worldwide. In Mexico, Fusarium boothii and F. avenaceum are the dominant species causing FHB of wheat (Cerón-Bustamante et al. 2018). During the 2017 to 2019 surveys, FHB symptoms were observed in wheat fields in the Highlands region of Mexico. Symptomatic spike samples were collected from 19 wheat fields in five states (Tlaxcala, Hidalgo, Puebla, Estado de México, and Morelos). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 95 monoconidial isolates were obtained. Morphological features of seven isolates were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pink pigment on the reverse side after 7 days of incubation at 25℃. On carnation leaf agar (CLA), macroconidia (n = 100) were hyaline, falcate, with 3 to 6 septa, measuring 25.2 to 43.1 × 2.8 to 5.1 μm, and foot-shaped basal cell. Chlamydospores were ellipsoidal or subglobose and produced in chains. These seven isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University under acc. nos. UACH428 to UACH434. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were amplified, and sequenced with the primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree, including published ITS, EF1-α, and RPB2 sequence data, was constructed for the Fusarium incarnatum-equiseti species complex (FIESC) based on Maximum Likelihood. Three species of the FIESC were identified into F. pernambucanum (five isolates), F. sulawesiense (one isolate), and F. clavum (one isolate). The sequences were deposited in GenBank with accession nos. OL347713 to OL347719 for ITS, OL365078 to OL365084 for EF1-α, and OL365072 to OL365077 for RPB2. The pathogenicity of the isolates was confirmed on wheat cv. Nana F2007 at the flowering stage in a glasshouse assay. The heads of 20 wheat plants were sprayed with a conidial suspension (1 × 105 spores/ml) of each isolate. Ten plants mock-inoculated with sterilized water served as the controls. All plants were placed in a moist chamber for 48 h. At 10 days after inoculation, typical FHB symptoms were visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected heads and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Previously, three isolates of Fusarium sp. belonging to the FIESC, were associated with FHB of wheat in Mexico (Cerón-Bustamante et al. 2018); however, this is the first report of F. pernambucanum, F. sulawesiense, and F. clavum causing FHB of wheat in Mexico and worldwide (Farr and Rossman 2021). Further studies should be focused on determining the distribution, prevalence, and toxigenic potential of the isolates of the FIESC associated with wheat diseases in Mexico.

The complete plastid genome of the endangered shrub Brassaiopsis angustifolia (Araliaceae): Comparative genetic and phylogenetic analysis.

Brassaiopsis angustifolia K.M. Feng belongs to the family Araliaceae, and is an endangered shrub species in southwest China. Despite the importance of this species, the plastid genome has not been sequenced and analyzed. In this study, the complete plastid genome of B. angustifolia was sequenced, analyzed, and compared to the eight species in the Araliaceae family. Our study reveals that the complete plastid genome of B. angustifolia is 156,534 bp long, with an overall GC content of 37.9%. The chloroplast genome (cp) encodes 133 genes, including 88 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. All protein-coding genes consisted of 21,582 codons. Among the nine species of Araliaceae, simple sequence repeats (SSRs) and five large repeat sequences were identified with total numbers ranging from 37 to 46 and 66 to 78, respectively. Five highly divergent regions were successfully identified that could be used as potential genetic markers of Brassaiopsis and Asian Palmate group. Phylogenetic analysis of 47 plastomes, representing 19 genera of Araliaceae and two related families, was performed to reconstruct highly supported relationships for the Araliaceae, which highlight four well-supported clades of the Hydrocotyle group, Greater Raukaua group, Aralia-Panax group, and Asian Palmate group. The genus Brassaiopsis can be divided into four groups using internal transcribed spacer (ITS) data. The results indicate that plastome and ITS data can contribute to investigations of the taxonomy, and phylogeny of B. angustifolia. This study provides a theoretical basis for species identification and future biological research on resources of the genus Brassaiopsis.