EF-1α Gene Research Articles

High Quality Rna Isolation and Exogenous Reference Gene for Real-time Pcr In Cedrela Odorata Seed Physiology Studies

Seed banks represent an important strategy for the conservation of forest genetic resources, although a basic understanding of the physiological changes that seeds undergo during storage that affect quality and germination is still lacking for most tropical and subtropical species. Here, we describe the optimisation of an RNA isolation procedure and reference gene normalisation for expression analysis in Cedrela odorata (cedro or Spanish cedar) seeds during different physiological states, as well as in the steady-state stem and leaf. The expression profiles of five endogenous candidate reference genes ( 18S , EF1α , GAPDH , CDC27B , PP2A2 ) and an exogenous ( HMBS ) gene were evaluated by using dedicated algorithms, including Genorm, Normfinder, Bestkeeper and Ct. We found that the expression of all endogenous genes varied considerably in response to both ageing and hydration. Therefore, using the external HMBS was a suitable alternative to evaluate gene expression in these highly contrasting physiological conditions. The reference genes EF1α and GAPDH were the most stable, and could be used for normalisation of qRT-PCR results under specific circumstances.

First report of Fusarium incarnatum-equiseti species complex as the causal agent of pod rot of black gram (Vigna mungo L.) in India.

Black gram (Vigna mungo L.) is important pulse crop and is the third-most cultivated pulse in India (Swaminathan et al. 2023). In August 2022, pod rot symptoms were observed on a black gram crop with 80 to 92% disease incidence in the Crop Research Center, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar (29.0222° N, 79.4908° E), Uttarakhand, India. Disease symptoms included white to salmon pink fungal-like growth over the pods. The symptoms were more severe initially at the tip of the pods, which expanded at later stages to cover the entire pod. Seeds present in the symptomatic pods were severely shrivelled and non-viable. Ten plants from the field were sampled to identify the causal agent. Symptomatic pods were cut into pieces, surface-disinfested with 70% ethanol for 1 min to reduce contaminants, rinsed with sterilized water three times, dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) amended with 30 mg/liter streptomycin sulphate. After 7 days of incubation at 25°C, three Fusarium-like (FUSEQ1, FUSEQ2, and FUSEQ3) isolates were purified by single spore transfer and subcultured on PDA. Fungal colonies on PDA were initially white to light pink, aerial, and floccose, and later on, colonies turned ochre yellowish to buff brown. After transfer to carnation leaf agar (Choi et al. 2014), isolates formed hyaline, 3 to 5 septate macroconidia, 20.4 to 55.6 × 3.0 to 5.0 µm (n = 50) with tapered, elongated apical cells and prominent foot-shaped basal cells. Chlamydospores were thick, intercalary, globose, and abundant in chains. No microconidia were observed. Based on morphological characters, the isolates were identified as belonging to the Fusarium incarnatum-equiseti species complex (FIESC) (Leslie and Summerell 2006). For molecular identification of the three isolates, total genomic DNA was extracted using the PureLink® Plant Total DNA Purification Kit (Invitrogen™, ThermoFisher Scientific, Waltham, Massachusetts, United States) and used for amplification and sequencing of a portion of the internal transcribed spacer (ITS) region, the translation elongation factor-1 alpha (EF-1α) gene, and the second largest subunit of RNA polymerase (RPB2) gene (White et al. 1990; O'Donnell 2000). The sequences were deposited in the GenBank database (ITS: OP784766, OP784777, and OP785092; EF-1α: OP802797, OP802798, and OP802799; RPB2: OP799667, OP799668, and OP799669). Polyphasic identification carried out in fusarium.org indicated 98.72 % and 100% similarity of FUSEQ1 and FUSEQ2 respectively to F. clavum and FUSEQ3 had 98.72 % similarity to F. ipomoeae. Both of the identified species are members of the FIESC (Xia et al. 2019). Pathogenicity tests were carried out on 45-day-old potted plants of V. mungo with seed pods, which were kept in a greenhouse. Plants were sprayed with 10 ml of a conidial suspension of each isolate (107 conidia/ml). Control plants were sprayed with sterile distilled water. The plants were covered with sterilized plastic bags after inoculation to maintain humidity and kept in a greenhouse at 25 ± 2°C. Within ten days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were symptomless. The pathogenicity test was repeated twice. Fungi consistently reisolated from the symptomatic pods were confirmed by morphological characterization and molecular assays as described above to belong to the FIESC, whereas no fungus was isolated from control pods. Fusarium spp. causing pod rot in green gram (V. radiata L.) has also been reported from India (Buttar et al. 2022). To our knowledge, this is the first report of FIESC as a causal agent of pod rot of V. mungo in India. The pathogen has the potential to cause considerable economic and production losses in black gram, and for that reason, disease management strategies should be implemented.

Isolation and identification of Metarhizium guizhouense Xct1 and its pathogenicity to Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundThe fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is one of the most destructive invasive pests causing serious damage to maize crops in China. At present, the use of chemical pesticides is the main method to prevent and control S. frugiperda. However, the excessive use of chemical pesticides not only causes toxicity in humans and animals but also pollutes the environment and causes public harm. In this context, the present study aimed to isolate, identify and evaluate the pathogenicity of the native strain of Metarhizium guizhouense against eggs, larvae and pupae of S. frugiperda under laboratory conditions.ResultsThe fungal isolate was identified as M. guizhouense (Xct1) based on its morphological and molecular characteristics by sequencing ITS, β-tubulin and EF1-α gene. Meanwhile, the fungal isolate was highly pathogenic to eggs of the S. frugiperda, at the highest concentration of 1 × 108 spores/ml, the cumulative egg hatching rate was only 8.33% at 10 days post exposure. After 10 days of exposure to the highest concentration of 1 × 108 spores/ml, the LC50 values for the first–fifth larval instars of S. frugiperda were 8.12 × 104, 1.11 × 105, 5.55 × 105, 3.87 × 106 and 1.43 × 107 spores/ml, respectively.ConclusionsThe pathogenicity of M. guizhouense Xct1 to S. frugiperda larvae gradually decreased with increasing the developmental stages. Metarhizium guizhouense Xct1 was more pathogenic to egg and younger larvae of S. frugiperda. This provides a basis for determining the optimal period of control.

First Report of Fusarium oxysporum f. sp. vasinfectum causing Fusarium Wilt of Cotton in Kansas, U.S.A.

Fusarium wilt is one of the most devastating diseases of cotton (Gossypium spp.). It is caused by the soil-borne pathogen Fusarium oxysporum Schlechtend. f. sp. vasinfectum (Atk.) Snyd. & Hans (Atkinson, 1892). To date, eight races of F. oxysporum f. sp. vasinfectum have been reported worldwide based on their diverse genetics and reactions to the host (Cianchetta, et al. 2015). Fusarium wilt symptoms appear at all developing stages. The fungus enters through the roots and colonizes the vascular system, leading to discoloration and wilting (Davis et al. 2006). During a survey in the July 2021 growing season, cotton plants showing typical wilting symptoms, stem discoloration, and root rot (Fig. 1A and B) were observed in two cotton fields in Sumner County, Kansas. In order to confirm the causal agent, root tissues and lower parts of the stems were collected from 25 diseased cotton plants and cleaned as previously described by Larren et al, (2001). Small segments (1-2 cm) of roots and stems were incubated on potato dextrose agar (PDA) at room temperature. The isolates were purified with the single spore method and 23 of 25 isolates (92%) showed typical morphology of F. oxysporum, with a white-peach pigmentation as reported previously (Leslie and Summerell 2008). Two isolates, CK13B and CK18A, one from each of the two fields were selected, and genomic DNA was extracted with E.Z.N.A Fungal DNA Mini kit (Omega Bio-tek, Norcross, GA). Polymerase chain reaction (PCR) assays were performed as reported previously (Ortiz, et al. 2017) to amplify and sequence a portion of three nuclear genes: translation elongation factor (EF-1α), phosphate: H* symporter (PHO), and β-tubulin (BT). BLASTn analysis of CK13B sequences showed 93-98% identity with F. oxysporum f. sp. vasinfectum isolates originating from Australia, with 93.43% (1706/1826 pb) identity of EF-1α to isolate AuSeed14 (KT323873), 95.11% (1849/1944 bp) of PHO to isolate AuK24232 (KT323909), and 97.56% (1840/1886 bp) identity of BT to isolate AusSeed14 (KT323833). Sequences of CK13B for EF1-α, PHO, and BT genes were submitted to GenBank, accession no. of ON754247, ON754248, and ON754249, respectively. The CK18A isolate showed high identity with F. oxysporum f. sp. vasinfectum races 1, 2, and 6 lineage isolates (Ortiz et al., 2017), with 99.89% (1822/1824 bp) identity of the EF1-α gene to isolate CDR238 (KT323838) from Arkansas, 98.36% (1916/1948 bp) identity of the PHO gene to isolate CDR1131 (KT323887) from Louisiana and 98.78% (1864/1887 bp) identity of the BT gene to isolate ATCC36198 (KT323799) from Brazil. Sequences of the CK18A isolate were submitted to GenBank, accession no. ON651444, ON725043, and ON725044, respectively. Pathogenicity of both isolates was tested on G. hirsutum as previously described (Kim et al. 2005) with three replicates per isolate and a control treatment in a growth chamber. Briefly, cotton seedlings were maintained with 12 h light/dark cycle at 25-20 °C. At the first true leaf stage seedlings were uprooted, roots were cleaned by rinsing with sterile water then placed in a conidial suspension (1×106 conidia/ml) for 2 h while the healthy control seedlings were dipped in water only. Plants were put into sterile soil, and the results were observed and recorded every seven days for a period of 30 days. The inoculated plants showed the same symptoms as those observed in the fields, wilting, leaf chlorosis, and necrosis. Both isolates were re-isolated from the roots of the inoculated plants. DNA was extracted and used for PCR with the specific gene primers as listed above. The sequences matched those of the original isolates, completing the Koch's pustulates. To our knowledge, this is the first report of F. oxysporum f. sp. vasinfectum causing Fusarium wilt disease of cotton in Kansas. These results will help growers select cultivars and design disease control strategies accordingly (Davis, et al., 2006). Further work is needed to determine the specific races in the area.

First report of Botryosphaeria dothidea as the causal agent of a new fruit rot disease of pepper in China.

Pepper is an important and widely cultivated economic vegetable in the world (Yin et al., 2021). In June 2021, approximately 25% to 33.3% of the pepper plants had rot disease symptoms in Zhuanghang Comprehensive Experimental Base (30.894829 °N, 121.391374 °E), Fengxian district, Shanghai city, China. Water-soaked spots appeared on fruits that increased in size and leading to smelly fruit decay. To isolate the pathogen, three pepper samples with severe symptoms were collected. The samples were surface disinfected with 70% ethanol for 30 sec, 10% chlorine bleach for 10 min, rinsing with sterile water for three times and the rot tissues were cut and dried on sterile filter paper. The dried paper was later placed on potato dextrose agar (PDA) medium and incubated at 28°C (Tang et al., 2021). After 2-3 days, four types of colonies with different colony appearances were observed, in which only one can induce fruit rot phenotype (data not shown). Four isolates were cultured for molecular identification in each type. ITS1/ITS4, T1/βt-2b and EF1-526F/EF1-1567R primers were used to amplify the internal transcribed spacer region (ITS), the beta-tubulin (TUB2) and the translation elongation factor I alpha (EF1-α) genes, respectively (Chen et al., 2018) and corresponding sequences from the isolates were analyzed with BLAST. Sequences of the isolate which can induce pepper decay were submitted to GenBank under the accession numbers of OM663701 (ITS), OM720127 (TUB2) and OM720128 (EF1-α). The results showed that the pathogen had 99% sequence homology to most strains of Botryosphaeria dothidea (B. dothidea) and displayed the highest sequence similarity to strain LBSX-1 (ITS: KF55123), strain JGT01 (TUB2: MW202404) and isolate CZA (EF1-α: MN025271). Based on molecular characterization, the isolate was identified as B. dothidea isolate SH01. A phylogenetic tree was constructed using Maximum Parsimony (MP) methods by MEGA7, and showed that SH01 was closely related to isolate CMW9075. To confirm the pathogenicity, five healthy pepper fruits were surface sterilized, 500μl of conidial suspension (1×103 conidia/ml) were injected into pepper (sterilized distilled water as control). Six days post inoculation (dpi), fruit rot symptoms appeared and the pepper decayed at 12 dpi. Four days post inoculation with mycelium plugs (from a 4-day-old culture on PDA, PDA plugs as control), hyphae were observed in the inoculation site and B. dothidea was re-isolated from the symptomatic areas, thus fulfilling Koch's postulates (Back et al., 2021, Chen et al., 2020). The pepper rotted severely at 7 dpi. The colonies of SH01 were pale to white and gradually turned into gray in 4-6 days. Conidia of the pathogen were unicellular, aseptate, hyaline and fusiform to fusoid, with dimensions of 19.7-23.5 μm × 3.8-5.2 μm (average = 21.9 μm × 4.8 μm, n = 50). Hyphae were transparent, branched and composed of multiple cells. The characteristic was consistent with the descriptions of B. dothidea (Vasic et al., 2013). B. dothidea belongs to Botryosphaeriaceae, causing widespread diseases in many plant species, commonly associated with cankers and dieback of woody plants and economic crops, such as plumcot trees (Back et al., 2021), eucalyptus (Yu et al., 2009) and soybeans (Chen et al., 2020) in China and Korea. Our findings reported for the first time that B. dothidea (SH01) can induce the pepper rot disease and future work on its pathogenesis may provide strategies for disease control against this fungus.

First report of Neofusicoccum australe causing dieback of honeybush in the Western Cape, South Africa.

Honeybush (Cyclopia spp.) is an indigenous, leguminous member of the Cape fynbos biome growing in the coastal winter rainfall districts of the Western and Eastern Cape Provinces of South Africa (Joubert et al. 2011). Honeybush is used for the production of herbal teas and is harvested from wild-growing and cultivated plantations (du Toit et al. 1998). Very little is known regarding diseases caused by pathogens on this indigenous plant. Only one report of twig dieback on honeybush caused by several Diaporthe Nitschke species have been reported in South Africa (Smit et al. 2021). Several honeybush producers reported poor growth and dieback in their C. subternata plantations in the Western Cape Province, South Africa. Symptoms included twig dieback, branch dieback, death of branches as well as death of entire plants. In April 2008, branches from 8-year-old cultivated plants with dieback symptoms were collected in Stellenbosch. Fungal isolations were carried out from affected material as described by Van Niekerk et al. (2004) which consistently revealed the presence of a Botryosphaeriaceae species. Two isolates were grown on water agar with sterile pine needles and incubated at 25˚C using a 12-hour day/night cycle and near-ultraviolet light. Pycnidia formed after two weeks. Morphological characteristics similar to Neofusicoccum australe (Slippers, Crous & Wingfield) Crous, Slippers & Phillips were observed (Phillips et al. 2013). Conidia were hyaline, aseptate, fusiform with subtruncate bases (16.8-)18.8-22.1(-24.6) × (4.8-)5.3-6.1(-6.4) µm (n=50). Conidiogenous cells were holoblastic, hyaline and subcylindrical to flask-shaped tapering to the apex (11-15 × 2 µm) (n=10). Colonies on potato dextrose agar were light primrose turning olivaceous grey after 7 days with a light-yellow pigment diffusing into the medium. Mycelia was moderately dense with an appressed centre mat. The identity of the isolates was further confirmed by sequencing the ribosomal RNA Internal Transcribed Spacer (ITS) and the elongation factor 1-alpha (EF-1α) gene regions using primer pairs ITS4-ITS5 (White et al. 1990) and EF1-728F-EF1-986R (Alves et al. 2008), respectively. Sequences had a 100% similarity to N. australe ex-type CMW6837 isolate (accessions AY339262 and AY339270) (Slippers et al. 2004). Two isolates (STEU6554 and STEU6557) were deposited in the culture collection at the Department of Plant Pathology at Stellenbosch University and the sequences were submitted to GenBank with accession numbers ON745603, ON745604, ON746573 and ON746574. Pathogenicity tests using the two N. australe isolates were conducted by inoculating two shoots each of three field-grown C. subternata plants with a 4mm colonised potato dextrose agar (PDA) mycelium plug of each isolate on wounds made by a 4mm cork borer (Van Niekerk et al. 2004). A third shoot was inoculated with a uncolonized PDA plug as the negative control. After 12 weeks, brown-black lesions that were significantly longer (average 55.2 mm) than the uncolonized agar plug control (16.1 mm) were observed. Lesions were observed in all three plants. Neofusicoccum australe was re-isolated (van Niekerk et al. 2004) from all inoculated shoots confirming Koch's postulates. The economic impact and damages caused by N. australe as well as its incidence and severity on honeybush in South Africa is unknown. However, the pathogen caused dieback of entire branches and death of plants indicating that it could be an important pathogen of honeybush. Additionally, N. australe is one of the most important disease-causing Botryosphaeriaceae pathogens on a wide range of economical fruit and vine crops globally (Mojeremane et al. 2020). This is the first report of N. australe as a known pathogen causing decline and dieback of C. subternata in South Africa.

The phylogeny and divergence time of Ophiocordyceps sinensis and its host insects based on elongation factor 1 alpha

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is endemic to the Qinghai-Tibet Plateau (QTP). The phylogeny and divergence time of O. sinensis and its host insects were analyzed for 137 individuals from 48 O. sinensis populations based on the elongation factor 1 alpha (EF-1α) gene. Lower nucleotide variation, with only 7 and 16 EF-1α haplotypes, was detected in O. sinensis and its host insects, respectively. The isolated and broad distribution patterns coexisted in both O. sinensis and its host insects on the QTP. The divergence time estimates show that O. sinensis and its host insects originated later than 14.33 million years (Myr) and earlier than 23.60 Myr in the Miocene period, and the major differentiation occurred later than 4 Myr. Their origin and differentiation match well with the second and third uplifts of the QTP, respectively. The host insects from the O. sinensis populations distributed around Qinghai Lake are inferredas an ancient and relict species that has survived various geological events of the QTP. It is suitable to estimate the divergence times of both O. sinensis and its host insects from the same individuals using one gene: EF-1α. Our findings of the origin, phylogeny, and evolution of the endemic species also support the epoch of geological events on the QTP.

First Report of Tobacco Root Rot Caused by Fusarium falciforme in China.

China is the largest producer of tobacco (Nicotiana tabacum L.) in the world with an estimated production of 2.4 million ton per year (Berbeć and Matyka 2020). In June 2021, a root disease was observed on tobacco in three surveyed counties (Xiangcheng, Linying and Jiaxian) in central Henan. Diseased plants exhibited leaf chlorosis and brown to purplish vascular discoloration of the taproot and lateral roots. Approximately 10 to 15% of the plants were symptomatic in the nine fields surveyed, representing 60 ha in total. Root segments (0.5 to 1 cm) from ten diseased plants were surface sterilized in 75% ethanol for 30 s followed by rinsing with sterile distilled water three times. Thirty air dried root pieces were placed on potato dextrose agar (PDA) and incubated at 25℃ in the dark for 2 days. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Yz01 to Yz10). Colonies on PDA showed abundant white to cream aerial mycelia with a yellowish-brown center on the reverse side after 7 days, and an average growth rate of 5 mm/day. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia had three to four septa, were falciform, with blunt apical cells and slightly hooked basal cell, and measured 20 to 41×3-6.5 μm (n=50). Spherical conidia clusters were formed at the apex of the conidiophores. Abundant reniform and cylindrical microconidia were one to two-celled, with apexes rounded, measuring 7 to 15×2 to 5 μm (n=50). The roughly spherical chlamydospores were intercalary or terminal, single or in chains, and rough walled. Such characteristics were consistent with the Fuarium solani species complex (FSSC) (Leslie and Summerell 2006). The translation elongation factor 1-alpha (EF1-α) gene of the ten cultures was amplified with primers EF1/EF2 (O'Donnell et al. 1998), and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequences of the ten cultures (Kumar et al. 2016). The RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes of the cultures were amplified with primers F5/G2R and RPB2F/R respectively (O'Donnell et al. 1998, 2010), and sequenced. The EF1-α, RPB1 and RPB2 sequences (GenBank accession nos. ON186742.1-ON186751.1, ON241133.1-ON241148.1, ON324054.1-ON324057.1) were 99.4 to 100% identical to the corresponding DNA sequences of Fusarium falciforme based on FUSARIUM-ID BLASTn analysis. Morphological and molecular results confirmed this species as F. falcifome (Díaz-Nájera et al. 2021; Velarde-Félix et al. 2022). Pathogenicity tests were performed in tobacco seedlings grown on autoclaved soil. Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with the representative culture Yz07 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7 to 14 days after inoculation for all the 90 inoculated seedlings. Fifteen diseased seedlings were randomly selected for tissue isolation, and F. falciforme was reisolated from the 15 seedlings and showed the same morphology and EF1-α gene sequence as the original isolate. Control plants remained asymptomatic and no pathogen was recovered. The results showed that F. falciforme can cause root rot of tobacco. F. falciforme was reported to cause tobacco wilt and root rot in Northwestern Argentina (Berruezo et al. 2018); however, this is the first report of F. falciforme causing root rot of tobacco in China. This species was previously reported in China affecting Weigela florida (Shen et al. 2019) and Dioscorea polystachya (Zhang et al. 2020), showing that F. falciforme has a broad host range in this country. These results may inform control tobacco root rot through improve crop rotations. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Outstanding Youth Science and Technology Fund Project of Henan Academy of Agricultural Sciences (2022YQ09) and Science and Technology Innovation Team project of Henan Academy of Agricultural Sciences (2022TD26).

First Report of Strawberry Wilt Caused by Fusarium oxysporum Schltdl. in Jordan.

Strawberries (Fragaria ananassa Duch.) are grown in Jordan year round due to the diversity of climatic conditions and the possibility of growing local or foreign varieties. More than six thousand greenhouses are planted with strawberries in the highlands and the Jordan Valley. About 12 thousand tons of strawberry are produced annually and 2000 tons are exported to European and Arab countries. In April and May 2019, symptoms of wiltand whole plant collapse were observed on approximately 30% of commercial strawberry cv. Deli gent crop in the Jordan Valley (Dayr Alla long.35.6188766, lat. 32.227465). Plants were either dead or showing symptoms including vascular wilt, external and internal discoloration of the stems, and dead shoots. Forty symptomatic plants were collected from 10 greenhouses, and stem fragments were surface sterilized and plated on potato dextrose agar (PDA). Six fungal isolates showed morphological characteristic of Fusarium oxysporum. Colonies on PDA were purple-violet, floccose, with abundant aerial mycelium; colony margins were irregular. Macroconidia were falcate, apical cells had a blunt or papillate shape, basal cells were foot shaped, three- to five-septate, hyaline, smooth, thin-walled, and 37 - 42 × 3 - 6.0 μm in diameter. Aerial microconidia were abundant, hyaline, ellipsoidal, zero to one-septate: 5 - 11 × 2 - 4.0 μm. Chlamydospores were globose to subglobose, intercalary or terminal, with an average diameter of 12 μm (Figure 1: A, B and C) (Nelson et al, 1983; Leslie and Summerell, 2006). Four representative isolates (FoSB2021JO-02, FoSB2021JO-06, FoSB2021JO-08 and FoSB2021JO-09) were DNA extracted, amplified with the translation elongation factor 1-α (EF1α) gene (EF1/EF2) primers (Geiser et al., 2004), and sequenced at Macrogen Inc, South Korea. Forward and reverse sequences were received, assembled and consensus sequence were produced using the BioEdit sequence alignment editor. Consensus sequences of the four isolates were used to conduct BLASTn queries of NCBI website (https://www.ncbi.nlm.nih.gov) and were 100%, 99.9%, 99.6%, 99.9% identical to F. oxysporum accessions MN417194.1, MK968948.1,MK968948.1, and MK968952.1, respectively. A phylogenetic tree with 1000 bootstraps was created using MEGA 7 software (Kumar et al. 2016) (Figure 2). Similarly, the four isolates were 99.5%, 100%, 100%, and 100% identical F. oxysporum reference accessions AF008507, FJ985275, FJ985275, and FJ985278 in the Fusarium MLST database, respectively. Consensus sequences of the four isolates were submitted to GenBank and accession numbers were assigned (OK040155 - OK040158). For pathogenicity tests on strawberries, a spore suspension of 1 × 105 conidia/ml was prepared separately for six isolates. Roots of identical 2-month-old healthy strawberry seedlings (15 plants of cv. Deli-gent) were cut and dipped in the spore suspensions for 30 min. They were then planted in 25 x 20 cm deep plastic pots filled with a sterile mixture of peat - moss, perlite, and vermiculite (60:20:20v/v). Control strawberry plants were soaked in water prior to planting. All plants were placed in a greenhouse at 25°C ±2 along with 15 uninoculated control plants. After 30 days, inoculated plants displayed similar symptoms to those observed in the green house, whereas control plants were symptomless. Roots from symptomatic plants were cultured on PDA and F. oxysporum was recovered and identified morphologically as F. oxysporum. To our knowledge, this is the first report of Fusarium wilt of strawberry in Jordan. The pathogen can cause significant economic losses to strawberries in Jordan and worldwide. Therefore, it is extremely important for disease control in nurseries to determine the infection source and possible factors that increase the incidence of infection to control the disease.

First Report of Top Blight of Cunninghamia lanceolata Caused by Diaporthe unshiuensis and Diaporthe hongkongensis in China.

Cunninghamia lanceolata (Lamb.) Hook. is an important conifer species widely planted in southern China. A top blight, with an incidence of 20% (40/200 seedlings), occurred on 1-year-old seedlings of C. lanceolata in a nursery, Luzhai, Guangxi, China in August 2021. The disease mainly occurred on shoot tips. The infected needles and shoots appeared brown to brownish red. White conidial tendrils oozed from pycnidia under wet-weather conditions. Lesion margins from fresh samples were cut into small pieces (n=100), which were sterilized according to Mao et al., and placed on potato dextrose agar (PDA) at 25°C. Three isolates (GXJ2, GXJ4, and GXJ6) were obtained and deposited in The China Forestry Culture Collection Center (CFCC 55717, CFCC 55716, and CFCC 55722). The colony of GXJ2 on PDA was white, with sparse aerial mycelia, and became grey with time. The α conidia were fusiform, hyaline, and aseptate, 6.7±0.6 μm × 2.6±0.2 μm (n=30). The β conidia were filiform, hyaline, and curved, 30.4±2.1 μm × 1.4±0.1 μm (n=30). Colonies of GXJ4 and GXJ6 were white, with moderate aerial mycelia, which collapsed at the center, and the collapsed parts were iron-gray. The α conidia were 7.8±0.8 μm × 2.5±0.2 μm (n=30). The β conidia were absent. Morphological characters of 3 isolates matched those of Diaporthe spp.. The partial sequences of ITS, EF1-α, CAL, β-tub, and HIS genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R and CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b according to Gomes et al. 2013, respectively. The sequences for the five genes of each of 3 isolates were deposited in GenBank (Accession Nos. see Supplementary Table 1). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of GXJ2 were highly similar (>99%) with sequences of Diaporthe unshiuensis, while sequences of GXJ4 and GXJ6 were highly similar (>99%) to those of D. hongkongensis (Supplementary Table 1). Phylogenetic analyses using concatenated sequences placed GXJ2 in the clade of D. unshiuensis, while GXJ4 and GXJ6 in the clade of D. hongkongensis. Based on the phylogeny and morphology, GXJ2 was identified as D. unshiuensis, GXJ4 and GXJ6 as D. hongkongensis. Pathogenicity tests were performed on nine 1-year-old seedlings of C. lanceolata, and 10 needles at shoot tip per seedling were slightly wounded and inoculated with 5-mm mycelial plugs from one of 3 isolates. Three control seedlings were treated with PDA plugs. Each plant was covered with a plastic bag after inoculation and kept in an air-conditioned nursery at 25°C/16°C (day/night). The symptoms appeared 5-8 days after inoculation and were similar to those observed in the nursery. D. unshiuensis and D. hongkongensis were re-isolated from the inoculated seedlings and were confirmed based on morphology and ITS sequences. The controls were symptomless, and no fungus was isolated from them. D. unshiuensis was first reported as an endophyte on the fruit of Citrus unshiu, and caused peach constriction canker, shoot blight of kiwifruit. D. hongkongensis was first described from fruit of Dichroa febrifuga and caused shoot canker of pear, shoot blight and leaf spot of kiwifruit, and fruit rot of peach. This is the first report of D. unshiuensis and D. hongkongensis causing the top blight of C. lanceolata. This study provides a basis for controlling this newly emerging disease in the nursery.

Stepwise Optimization of the RT-qPCR Protocol and the Evaluation of Housekeeping Genes in Pears (Pyrus bretschneideri) under Various Hormone Treatments and Stresses

Real-time quantitative PCR (RT-qPCR) has become a widely used method for exploring plant gene expression level. The method requires using some stably expressed genes as a reference to accurately normalize the RT-qPCR data. However, under various stresses and hormone treatments, the levels of most reference genes vary. Environmental variations also influence their expression levels. The lack of validated, stably expressed reference genes can mislead the study of gene function in pears. “Huangguan” pears have recently become the focus of research on stress resistance mechanisms, such as high resistance. Therefore, the aim of this study was to select the optimal reference genes in Huangguan pears, and we analyzed the expression of the genes EF1α, ACT, SKD1, YLS8, UBQ, GAPDH, TUB, and WDP in a series of pear leaf sets under various stresses and hormone treatments. Using different statistical algorithms, we found that under various treatments, the WDP gene had more stable expression, ACT was the most stable under MeJA treatment, YLS8 was the most valuable reference gene under ABA hormone and heat stress conditions, and GAPDH showed worst results compared to other housekeeping genes, except under heat stress. These results will supply valuable and updated information for the selection of housekeeping genes in pears under biotic and abiotic stresses in the future.

Occurrence of root rot caused by Fusarium fujikuroi and Fusarium proliferatum on peanut in China.

Root rot of peanut, caused by Fusarium spp., is a devastating disease in most peanut cultivation regions. In this reported outbreak, Fusarium root rot of peanut has been observed in Henan province, China in July 2021. About 20% of peanuts in a field (0.66 ha) were affected. Early symptoms comprised the wilt of the lower leaves, and the darkening of the vascular tissue of roots, which turned brown. Progressively, the whole plant wilted, the roots rotted, and the plant ultimately died. Pathogenic species were isolated from plants showing symptoms of root rot in the field. A total of 206 Fusarium isolates were generated, and 16 isolates were preliminarily identified as Fusarium fujikuroi based on morphological characteristics. Isolates were obtained and grown on PDA plates. Isolates developed floccose white aerial mycelia with reddish-pink coloration in the medium in 2 weeks on the benchtop. Macroconidia were 3-5 septate, measuring 27.5 to 48.8 × 2.6 to 3.8 μm (avg. 36.7 × 3.6 μm, n=50). Microconidia were abundant in chains, mainly asepatate, oval to kidney-shaped, 4.0 to 11.6 × 2.5 to 4.1 μm (avg. 5.8 ×3.1 μm, n=50). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al.1990) (Genbank assessions MZ831304 to MZ831308), the partial calmodulin gene (CAM, primer CL1/CL2A, O'Donnell.)(Genbank assession MZ856333 to MZ856337) and the partial translation elongation factor (EF-1α) using primer EF1/EF2 (Geiser et al.)(Genbank assession MZ856338 to MZ8564342). FUSARIUM-ID analysis showed 98.18% to 100% similarity with sequences of the F. fujikuroi species complex. The phylogenetic analysis was conducted using a neighbor-joining algorithm based on the ITS, CAM, and EF-1α gene sequences. The isolates were clustered with F. fujikuroi clade (Supplementary Fig.1). Koch's postulates were conducted using a sand-cornmeal-inoculum-layer method (Bilgi et al.). Briefly, 400 ml plastic boxes were filled with 15g of sterilized premium-grade coarse vermiculite, followed by a 15 g of inoculum prepared as sand-cornmeal mixture inoculum. The inoculum for each F. fujikuroi isolates was prepared by infesting a pre-sterilized sand-cornmeal mixture with three 5 mm plugs of cultures. Three F. fujikuroi isolates and PDA plugs were inoculated to serve as positive control and non-inoculated control treatments, respectively. The completed colonization of the sand-cornmeal mixture was finished by incubating at 25 ℃ for 7-10 days. Eight pre-germinated seeds of cv. Luhua No.1 was then covered with another 8 g of vermiculite. Peanuts were grown at 25 °C with 85% relative humidity under a light/dark cycle of 14h/10h. After 14 days of incubation, the inoculated plants showed typical symptoms of root rot similar to those in the field: pre-emergence damping-off, reddish-brown lesions on the tap, and lateral roots. F. fujikuroi was successfully re-isolated from inoculated plants but not from the controls and identified as described above. F. fujikuroi was reported to cause bakanae disease of rice (Amatulli et. al.), and root rot of soybeans (Zhao et. al.). To the best of our knowledge, this is the first record of F. fujikuroi causing root rot of peanut in China.

First Report of stem rot in Cymbidium sinense Caused by Fusarium oxysporum (FOSC) in China.

Cymbidium sinense (Jackson ex Andr.) Willd is a perennial terrestrial plant in the orchid family mainly distributed in China, Japan, India and Southeast Asia that occupies a strong position in the flower market due to its bright green leaves and fragrant flowers (Zhang et al. 2013). Cymbidium sinense is not only valued by people for its ornamental and economic value, but its roots have antiasthmatic medicinal properties (Ke et al. 2004). In August 2020, about 15% stem rot on two-year old C. sinense with varying severity was observed in five nursery gardens located in Enshi city (N 30° 16', E 109° 29'), Hubei province, China. Typical symptoms of C. sinense included roots and inner part of the pseudobulbs changing from white to brown and rotting. Leaves became brown and withered from bottom to top, and there was an obvious blight yellow halo at the junction of diseased and healthy tissue, which eventually caused the whole plant to wilt and die (Fig. 1d). To isolate the pathogen, a total of 15 leaf tissues from the disease-health junction (3 × 3 mm) from 5 individual plants (3 leaves/plant) with symptoms were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 3 min. The sterilized tissue was rinsed three times with sterilized water, and then placed on potato dextrose agar (PDA) for incubation at 28°C in the dark for 5 days. Isolated colonies were subcultured by a hyphal tip protocol. Thirteen fungal isolates were obtained. Through preliminary pathogenicity tests, we found that ten isolates induced leaf blight. These ten isolates with pathogenicity showed similar morphological characteristics, with initial white-flocculent aerial mycelium that secreted a lavender pigment and produced colonies with an irregular edge after 3 days on PDA. The ten strains were cultured on PDA plates at 28℃ for 5 and 15 days to observe colony and conidial characteristics. The ten strains were identified as Fusarium based on morphological characteristics (Leslie and Summerell 2006). Strain ML0303 was selected for further identification. Macroconidia were falciform, hyaline, slightly pointed at both ends with two to four septa, 24.0 ± 5.6 µm × 4.7 ± 0.8 µm (n = 50). Microconidia were hyaline, oval, globose, with zero to one septum, 5.5 ± 1.3 µm × 2.2 ± 0.5 µm (n = 50) (Fig. 1c). Total genomic DNA of strain ML0303 was extracted with a CTAB protocol (Stenglein and Balatti 2006). The translation elongation factor (EF-1α), RNA polymerase II second largest subunit (RPB2) and β-tubulin (Tub2) genes were amplified respectively using primer pairs EF1/EF2, RPB2-5F2/RPB2-7cR and T1/T22 respectively (O'Donnell. et al. 2010, O'Donnell. et al. 1997). The EF-1α, RPB2 and Tub2 (accession numbers-MW719874, OL614838, OL689398, respectively) gene sequences were submitted to GenBank. EF-1α, RPB2 and Tub2 sequences of ML0303 showed 99.5% - 100% identity respectively with Fusarium oxysporum in the Genbank and FUSARIUM-ID databases. The multilocus sequence data was used to infer a phylogenetic tree via a Neighbor-joining (NJ), Maximum-likelihood (ML) and Maximum-Parsimony(MP) together with reference sequences from GenBank. The topology of the three trees was similar; only the NJ tree is presented here. Strain ML0303 and F. oxysporum formed a clade supported with high values (NJ/ML/MP: 96,95,97). The results indicated that the fungus was F. oxysporum based on the phylogenetic analysis and BLASTn queries. For pathogenicity tests, conidia of strain ML0303 were collected by rinsing PDA plates. Two-year-old C. sinense grown in plastic pots filled with sterilized autoclaved sandy loam soil were used for the tests. Three pots (two plants/pot) were included in each treatment. Spore suspensions (106spores/ml) of strain ML0303 were used to irrigate the stem-zone of the plants, and sterile water was used as control. The two treatments were placed in a greenhouse and incubated at 28±2℃ with a 14-hour light/10-hour dark cycle. The experiment was repeated twice. After three weeks, stem rot symptoms were observed on C. sinense inoculated with ML0303, that were the as same as observed in the nursery (Fig. 1e-h). No symptoms were observed on the negative control. Fusarium oxysporum was re-isolated from the infected plants to fulfill Koch's postulates. Partial EF-1α and RPB2 gene sequences were used for molecular identification. Members of the FOSC are notorious for causing many diseases, which includes stem rot of Sulcorebutia heliosa and root rot of Torreya grandis (Garibaldi et al. 2020; Zhang et al. 2016). To our knowledge, this is the first report of stem rot by F. oxysporum on C. sinense in China. The finding of this pathogen provides a clear target for stem rot control.

First Report of Tobacco Fusarium Root Rot Caused by Fusarium redolens in China

First Report of Tobacco Fusarium Root Rot Caused by <i>Fusarium redolens</i> in China

Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

First Report of Mango malformation disease caused by Fusarium proliferatum in Mexico.

Mango (Mangifera indica L.) is the most economically important fruit in the tropical and subtropical regions of the world. Mexico is ranked the fourth largest mango producer worldwide with an approximate production of 2 396 675 t in 2019 (FAO 2020). Sinaloa is the principal mango production state in Mexico with 410,147 t in 2020 (SIAP 2021). Mango malformation disease (MMD) is one of the main limitations in the production of this crop worldwide, causing serious losses in yield. During December 2017 to April 2018, symptoms of MMD were observed in commercial mango in the municipality of El Rosario (Sinaloa, Mexico). These symptoms included malformed and compacted inflorescences, abnormal development of vegetative shoots with shortened internodes at an incidence of 25 %. Tissue from 15 symptomatic trees were superficially disinfested with 2% sodium hypochlorite and transferred to potato dextrose agar (PDA). Typical Fusarium spp. colonies were obtained from all samples. Fifteen pure cultures were obtained by single spore culturing. White to cream-colored aerial mycelia of typical Fusarium colonies were observed from all samples on PDA (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia (n = 50) were hyaline, relatively slender with a curve, 4 to 5 septate, and measured 39.5 to 76.8 x 5.7 to 9.5 μm. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 8.1 to 10.6 x 5.1 to 6.9 μm. Chlamydospores were observed. The EF1-α gene (O'Donnell et al. 1998) was amplified by PCR and sequenced from the isolates. The EF1-α sequence from one representative isolate (128FRSIN) was deposited in GenBank with the accession number MK932806. Maximum likelihood analysis was carried out using the representative EF1-α sequence for F. proliferatum (MK932806) and other Fusarium species. Phylogenetic analysis revealed the isolate most closely related was F. proliferatum (100% bootstrap). The molecular identification was also confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. The pathogenicity tests were carried out on healthy three-month-old mango plants. Twenty plants and five shoots per plant were inoculated with 20 µl of the conidial suspension (1 x 106 conidia/ml) (Freeman et al. 1999). Twenty plants served as noninoculated controls. Plants were maintained for 365 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Symptoms of multiple vegetative shoots and shortened internodes were observed four months after inoculation on the infected plants with an average disease of 4.5 in the first trial and 4.4 in the second assay according to the disease severity scale outlined by Iqbal et al., (2006). No symptoms were observed on non inoculated control plants after 365 days. One isolate per plant was isolated again from the plants with malformation symptoms (n=20), and identified again as F. proliferatum, by morphological and molecular characteristics. F. proliferatum was identified as the causal agent of MMD in China by Zhan et al. (2010). To our knowledge, this is the first report of F. proliferatum causing MMD in Mexico. The development of management strategies to prevent crop loss is required in this important mango production area.

First report of Lasiodiplodia theobromae causing leaf rot on Agave angustifolia in Mexico.

The agave crop (Agave angustifolia), is of economic importance for Mexico, for the agave is made mainly an alcoholic beverage called locally mezcal. In the state of Guerrero, in the municipality of Huitzuco de los Figueroa (18.2510026N, 99.2320182W, 1196 m above sea level), a severe disease affecting agave leaves was detected. The field symptoms consisted of pale to brown dark descending lesions, covering >50% of the leaf surface, in which the presence of pycnidia was observed. In an estimated area of 0.5 ha, the estimated incidence was 67% (n=100 plants). Symptomatic fragments from leaves (approximately 0.5 cm) were taken, superficially disinfected with 1% NaClO, and rinsed twice with sterile distilled water. Then they were transferred to potato dextrose agar (PDA) medium, and incubated at 28 °C. After five days, twelve representative isolates were selected and purified by the hyphal tip technique. In the PDA medium, the colonies were initially light gray, later they became dark, and after 22 days of incubation, the development of numerous dark pycnidia was observed on the surface of the medium. Initially, immature hyaline conidia, unicellular, oval, and double-walled were observed. The mature conidia were dark brown, oval, with one septum and longitudinal striation, and measured 17.5 to 27 [average 25.3 µm; n=50] × 10.5 to 15.7 [average 13.9 µm; n=50]. Based on the morphological characteristics, the fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). Isolates LAS3 and LAS4 were used for molecular identification, this was done by amplifying the regio internal transcribed spacer (ITS) of rDNA with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1-alpha ( EF-1α) genes using primers EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (LAS3; ON391564 and LAS4; ON391565 for ITS, and LAS3; ON368190 and LAS4; ON368191 for EF-1α). BLASTn analysis sequences of isolated LAS3 and LAS4 revealed for ITS 98.6% identity with L. theobromae (MK934699.1), and for EF-1α indicated 100% identity (MF422024.1). From concatenated sequences ITS-EF-1α regions, a phylogenetic analysis was carried out in MEGA X software, using the Maximum Likelihood and Kimura 2-parameter model with 1,000 bootstraps replicated; isolates LAS3 and LAS4 were clustered in the clade of the members of L. theobromae strains CAA006 (Alves et al. 2006), and INTA-IMC 1601 (Perez et al. 2018). The pathogenicity tests were carried out on 10 healthy 3 year-old agave plants, in which the mycelium of the LAS4 isolate was inserted at three equidistant points/leaf, using a sterile toothpick. Five healthy agave plants were inoculated only with sterile PDA as control treatment. The inoculated plants were covered with transparent plastic bags and housed in a greenhouse at 28 °C. After seven days, similar symptoms to those observed in the field were observed in all inoculated plants. Control plants did not develop symptoms. The fungus L. theobromae was re-isolated again from the infected leaves, fulfilling Koch's postulates. In China, L. theobromae has been reported as the cause of leaf rot on A. sisalana (Xie et al. 2016). To our knowledge, this is the first report of L. theobromae causing leaf rot on A. angustifolia in Mexico. This research is useful to design management strategies for leaf rot disease for local farmers of A. angustifolia.

First report of Diplodia mutila associated with Botryosphaeria canker and dieback of apple trees in Xinjiang, China.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

Phylogenetic evidence for a clade of tick-associated trypanosomes

BackgroundTrypanosomes are protozoan parasites of vertebrates that are of medical and veterinary concern. A variety of blood-feeding invertebrates have been identified as vectors, but the role of ticks in trypanosome transmission remains unclear.MethodsIn this study, we undertook extensive molecular screening for the presence and genetic diversity of trypanosomes in field ticks.ResultsExamination of 1089 specimens belonging to 28 tick species from Europe and South America led to the identification of two new trypanosome strains. The prevalence may be as high as 4% in tick species such as the castor bean tick Ixodes ricinus, but we found no evidence of transovarial transmission. Further phylogenetic analyses based on 18S rRNA, EF1-α, hsp60 and hsp85 gene sequences revealed that different tick species, originating from different continents, often harbour phylogenetically related trypanosome strains and species. Most tick-associated trypanosomes cluster in a monophyletic clade, the Trypanosoma pestanai clade, distinct from clades of trypanosomes associated with transmission by other blood-feeding invertebrates.ConclusionsThese observations suggest that ticks may be specific arthropod hosts for trypanosomes of the T. pestanai clade. Phylogenetic analyses provide further evidence that ticks may transmit these trypanosomes to a diversity of mammal species (including placental and marsupial species) on most continents.Graphical

First report of Lasiodiplodia theobromae associated with panicle blight of grapes (Vitis vinifera L.) in India.

Grape (Vitis vinefera L.) is a popular horticulture crop in Karnataka, India. A fungal pathogen caused panicle blight on panicles with immature fruit and severity increased subsequently in the grape growing regions of Devanahalli and Doddaballapur, Karnataka, between August and September 2019. The disease incidence varied from 15 to 18 percent in around 45 hectares of grape vineyards surveyed. The associated fungal pathogen was isolated on Potato Dextrose Agar (PDA) medium (HiMedia Laboratory, Mumbai, India) amended with Chloramphenicol. A total of 12 fungal isolates were obtained and identified based on morphology. Fungal cultures obtained from all the panicle blight affected samples were fluffy grayish to black with profuse, dense mycelium. Microscopic examinations revealed that the conidia ellipsoidal, two celled and hyaline when young, and developed dark brown pigments at maturity. Mature conidia measured 18.24±2.35 to 26.62±3.11 μm long and 10.32±1.08 to 12.57±1.82 μm width (n=30). The fungal pathogen was identified as a Lasiodiplodia sp. based on colony morphology and microscopic features. A total of three representative isolates L. theobromae (Vv12, Vv15, and Vv19) were selected for molecular identification based on ITS-rDNA, tub2 and EF-1α gene sequences and phylogenetic analysis. Genomic DNA was isolated from 12 day old cultures and ITS-rDNA, tub2 and EF-1α genes were amplified using ITS1/ITS4; Bt2a/Bt2b and EF1-728F/986R primer pairs, respectively (White et al., 1990; Glass and Donaldson, 1995, Carbone and Kohn, 1999). PCR amplicons were sequenced and the sequences were deposited in GenBank with the accession number ITS: MZ855866.1; MZ855867.1; MZ855868.1; tub2: MZ868708.1; MZ868709.1; MZ868710.1 and EF-1α: OM604750; OM604751; OM604752 respectively. The phylogeny was constructed based on combined ITS, EF-1α and the tub2 regions. Maximum Likelihood (ML) analysis was conducted and an ML tree was constructed with the substitution models (branch support was evaluated by 1,000 bootstrap replications). Combined phylogeny confirmed that the sequences shared a common clade with L. theobromae. Based on micro-morphological features and multi-locus sequence phylogeny, the associated fungal pathogen was identified as L. theobromae. There are no reports on the occurrence of L. theobromae causing panicle blight on grapes from India. Further, the pathogens association was confirmed through pathogenicity assay conducted on field harvested healthy bunches of grapes maintained under humid chamber. A total of 10 grape bunches were inoculated with a mycelial disc on the rachis of the panicle and incubated in a moist chamber for 5 days and control sets were inoculated with only agar plugs. The experiments were conducted in three replicates and repeated twice. A total of 21 panicle bunches developed typical rot symptoms 12-days post inoculation. The identity of the pathogen was confirmed based on micromorphology and cultural features after re-isolation (n=5), thus proving the Koch postulates and confirming the association of L. theobromae with panicle blight of grapes. Lasiodiplodia species are known to cause dieback, stem blight, leaf blights and spots on various crop plants. Mathur (1979) mentioned the occurrence of L. theobromae on grapes, however, no further details are available on the part associated, as well as morphological and molecular confirmation of L. theobromae. This is the first report of the L. theobromae causing panicle blight disease of grapes in India. Further, understanding the host range for L. theobromae and its variation will help to draw suitable disease management strategies.