Brown Center Research Articles

Infundibulicybe swatica (Basidiomycota, Agaricales), a new species from Pakistan

A new species, Infundibulicybe swatica is described and illustrated from District Swat, Khyber Pakhtunkhwa, Pakistan. The new taxon is characterized by pale yellow to pale yellow brown to brown pileus with a dark brown center; a bicolorous stipe, white at the apex and pale yellow brown hues at the base; hyaline, lacrymoid to sub-lacrymoid, small, inamyloid basidiospores (6.5 × 4.2 µm); and the absence of hymenial cystidia. Molecular phylogenetic analyses based on nuclear ribosomal Internal Transcribed Spacer (ITS) and nuclear ribosomal Large Subunit (LSU) regions confirm the novelty of the taxon.

The Potential for Hyperspectral Imaging and Machine Learning to Classify Internal Quality Defects in Macadamia Nuts

Tree nuts are rich in nutrients, and global production and consumption have doubled during the last decade. However, nuts have a range of quality defects that must be detected and removed during post-harvest processing. Tree nuts can develop hidden internal discoloration, and current sorting methods are prone to subjectivity and human error. Therefore, non-destructive, real-time methods to evaluate internal nut quality are needed. This study explored the potential for VNIR (400–1000 nm) hyperspectral imaging to classify brown center disorder in macadamias. This study compared the accuracy of classifiers developed using images of kernels imaged in face-up and face-down orientations. Classification accuracy was excellent using face-up (>97.9%) and face-down (>94%) images using ensemble and linear discriminate models before and after wavelength selection. Combining images to form a pooled dataset also provided high accuracy (>90%) using artificial neural network and support vector machine models. Overall, HSI has great potential for commercial application in nut processing to detect internal brown centers using images of the outside kernel surface in the VNIR range. This technology will allow rapid and non-destructive evaluation of intact nut products that can then be marketed as a high-quality, defect-free product, compared with traditional methods that rely heavily on representative sub-sampling.

First Report of Neopestalotiopsis spp. Causing Leaf Spot and Petiole Blight on Strawberry in New England.

In October 2023, a Connecticut grower contacted The Connecticut Agricultural Experiment Station about a field of strawberry plants (Fragaria × ananassa) (cv. Ruby June) showing symptoms of severe leaf spotting and visual wilting. Upon visiting the field, leaves had lesions with a diffuse black halo and a light brown center and wilting symptoms, which appeared driven by petiole lesions and presented as dark brown stripes with a reddish-purple halo. Symptoms were observed on 80 to 90% of plants within the block, nearly all of which (>90%) presented with both leaf spots and severe wilting. Diseased tissue was collected from 20 leaves and 25 petioles, sterilized in 0.6% NaOCL, and plated on potato dextrose agar. After hyphal tipping a morphologically identical fungus was isolated from 70% of leaves and 88% of petioles, which formed a dense white mycelial mat with moderate aerial mycelium and conidiomata that exuded dark brown conidial masses. The underside of the mycelial mat was yellowish. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate with a single basal appendage and 2-5 apical, matching the description of species within the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The average conidia (n=74) length, not including appendages, was 29.9 ± 2.1 µm and the average width, at the widest point, was 7.5 ± 0.7 µm. Aerial hyphae were collected from two isolates, CT58-1 and CT62-2, and DNA was extracted for further molecular characterization. PCR was performed with primers targeting actin (ACT), β-tubulin (TUB2), and ITS prior to amplicon sequencing (Carbone and Kohn 1999; Hassan et al. 2018). Sequences were queried against the NCBI whole genome shotgun database, and aligned sequences from 13 species (including Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis) were collected for each locus. Sequences were aligned, trimmed, and concatenated using Mega11, and IQ-TREE was employed for model selection (Nguyen et al. 2015; Tamura et al. 2021). A maximum-likelihood tree placed the isolates in a high-confidence cluster with Neopestalotiopsis rosae, confirming this placement of these isolates within the genus (CT58-1 Accession #: PP715979-89; PP707735). To confirm pathogenicity, CT58-1 was grown on autoclaved strawberry leaves to induce sporulation, and a suspension of 105 spores/ml was made. Five milliliters of this spore suspension was sprayed on six 6-week-old strawberries (cv. Jewel), and water was sprayed on the same number of control plants. Plants were at 100% humidity for two days and then kept in the greenhouse for 3 weeks to observe symptoms. Inoculated plants presented with identical leaf spot and petiole lesions to field samples and no visual symptoms were observed on control plants. New isolations were made from infected petioles, which produced morphologically identical spores to those described above, and ITS/ACT loci sequencing yielded sequences identical to those of CT58-1. Spore production and plant inoculations were repeated with this new isolate, and identical symptoms were observed. This is the first report of Neopestalotiopsis infecting strawberries in New England and given the high disease incidence in the initial infected field and relative lack of disease in a neighboring field, it is likely that this pathogen was introduced on bare root plants. As the plants were sourced from a nursery in Ontario, Canada, it is likely that the pathogen is capable of overwintering in the Northeastern United States.

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

Hermatomyces pyriformis sp. nov., a novel dematiaceous hyphomycete (Hermatomycetaceae, Pleosporales) associated with medicinal plants in Yunnan Province, China

During investigations of hyphomycetous fungi from medicinal plants in Yunnan Province, China, a novel species of Hermatomyces was discovered. Hermatomyces pyriformis is introduced based on morphology and phylogenetic analyses of combined LSU, ITS, tef1-α and rpb2 sequence data. This fungus is characterized by having sporodochial conidiomata and determinate, hyaline, pyriform conidiogenous cells bearing brown, globose to subglobose or ellipsoidal, muriform lenticular conidia with dark brown center and brown peripheral cells. Phylogenetic results indicated that two isolates of H. pyriformis represent a distinct lineage and can be recognized as a new species. A detailed description and illustration are provided for the new species.

Characterization and pathogenicity of Alternaria species associated with leaf spot on Plantago lanceolata in Sichuan Province, China

AbstractPlantago lanceolata has been increasingly cultivated in recent years as a forage crop due to its high feeding quality, good adaptability and medicinal benefits. Between 2015 and 2023, Alternaria leaf spot (ALS), appearing as reddish‐brown necrotic spots with a greyish white to pale brown centre, was commonly observed in two forage germplasm nurseries of P. lanceolata of Sichuan Province, China. Initially, 139 Alternaria strains were isolated from leaf lesions, and then identified using the identity of rDNA internal transcribed spacer (ITS) sequences in the NCBI database and the similarity of morphological characteristics. By pure culture technique, six representative isolates were finally obtained for the present study. Based on both multilocus phylogeny (SSU, LSU, ITS, gapdh, rpb2, tef1, endoPG, OPA10‐2, alt a 1, KOG1058 and KOG1077) and morphology, four Alternaria species were identified: three new records of A. alternata, A. arborescens species complex and A. gossypina on this host in China, belonging to section Alternaria, and a new species of section Porri, namely, A. lanceolatae sp. nov. Pathogenicity tests by inoculation on both detached leaves and whole plants revealed that A. lanceolatae was the most important causal agent of ALS, while the other three species were nonpathogenic to P. lanceolata. This study characterized a novel pathogenic Alternaria species of section Porri from P. lanceolata in China and could contribute as useful research for possible ALS management.

New species, new records and common species of Pluteussect.Celluloderma from northern China.

Wood-rotting fungi are organisms that can decompose wood substrates and extract nutrients from them to support their growth. They play a crucial role in the material cycle of forest ecosystems. The genus Pluteus plays a significant role in wood decomposition. In this study, the morphology and molecular systematics of the sect. Celluloderma of the genus Pluteus were carried out. Pluteusbrunneodiscus was identified as a new species, along with the discovery of two new records, P.cystidiosus and P.chrysophlebius, and a common species, P.romellii. Pluteusbrunneodiscus is characterized by the brown center of the pileus that transitions to white towards the margins, with the surface cracking to form irregular granules. It is typically found in Populus forests growing on decomposing twigs or wood chips. Line drawings, color photographs, and phylogenetic analyses of related species within the genus Pluteus accompany the descriptions of these four species. The analyses are based on ITS + TEF1-α sequence data. Finally, a key for the twenty species within the sect. Celluloderma of the genus Pluteus, which has been documented in China, is provided.

Understanding chemical pathways of brown centre formation in laboratory induced and conventionally dried nut-in-shell macadamia kernels

World tree nut production has increased rapidly by around 50 % in the past decade; however, nut defects cause losses. For example, we know that brown centres are a major internal discolouration defect in macadamia nuts and are linked to the storage of nut-in-shell under improper conditions at high temperature and humidity. However, key chemical changes in brown centre kernels have not been described. In this study, we compared brown centres and white kernels from: 1) samples that were “induced” in the laboratory by storing at high moisture concentration; and 2) samples that were dried immediately after harvest using industry best practice methods recommended by the Australian Macadamia Society (AMS). We measured the moisture concentration, sugar concentration, fatty acid concentration, peroxide value, nutrient concentration and volatile compounds of induced and AMS samples. Our results showed that storing nut-in-shell macadamia under wet and hot conditions increased brown centres compared with samples immediately dried using the AMS regime, 10.33 % vs 1.44 %, respectively. Induced brown centres had significantly higher moisture concentrations than induced white centres. Volatile compounds including nonanoic acid, octanoic acid and 2,3 butanediol were identified and associated with brown centre formation in macadamia kernels and the initiation of lipid oxidation. Our results suggest sugar hydrolysis and the Maillard reaction are associated with brown centres both in laboratory induced samples and those formed using industry best practice drying methods. Our study suggests improper drying and storage at high temperature and high humidity are likely to result in brown centre formation. We recommend brown centre losses can be reduced by appropriate drying and storage practices.

First report of Diaporthe eres causing leaf spot on Rhododendron latoucheae Franch. in China.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

First Report of Leaf Blight of Hedychium coronarium Caused by Alternaria alternata in China.

Hedychium coronarium is an economically significant crop that is widely cultivated for its ornamental, aromatic, and medicinal value (Abbas et al. 2021). From 2020 to 2023, a leaf blight was observed in about 85% of H. coronarium growing in a production field (approximately 500 m2) at Southwest University, Chongqing, China (29° 150'-29° 41' N, 105° 17'-105° 44' E). Symptoms included dark brown necrotic tissue with a clear yellow border. When the disease became severe, affected leaves became dry and abscised. Symptomatic pieces (2 to 5 mm2) between necrotic and healthy tissues were collected from 20 leaf samples, then were immersed in 70% ethanol for 10 s, 0.1% mercury bichloride for 3 min, rinsed in sterile water three times, and placed onto potato dextrose agar (PDA). Four Alternaria isolates were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Cultures on PDA were initially white mycelium on the rim with a light brown center. At around the fourth day, the colony margin changed into light gray and the central part turned sooty black. Conidiophores were branched. Conidia were dark brown, ovoid or ellipsoid in shape, 3.4 to 13.2 μm × 4.1 to 23.5 μm (n = 50) with zero to four transverse and longitudinal septa. For molecular identification, DNA was extracted using the PlantGen DNA Kit CW0553A (Cwbio, Taizhou, China) for PCR amplification of internal transcribed spacer (ITS) region, and 28S large subunit ribosomal RNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen (Alt a 1) and actin (ACT) genes (Choi et al. 2022; Xie et al. 2022; Zhang et al. 2021). BLASTn searches showed that ITS, LSU, GAPDH, Alt a 1 and ACT of four isolates had 100% homology with the corresponding sequences of A. alternata strains MZ578214, KP940477, MK903028, MN655781 and MF564199, respectively. Representative sequences of one strain (AH1) with accession numbers of OK639009, OK639186, OK664976, OK664977 and OK664978 for ITS, LSU, GAPDH, Alt a 1 and ACT regions were deposited in GenBank. The maximum-likelihood tree generated by MEGA 5.10 demonstrated that the pathogenic isolate AH1 obtained from H. coronarium leaf was grouped in the same clade with A. alternata strain CBS121348, which was supported by 100% bootstrap values. To fulfill Koch's postulates, conidia were collected from a 7-day-old culture, suspended in sterile distilled water, and adjusted to 1 × 106 conidia/mL. Leaves on 6-month-old H. coronarium were surface disinfected with 1% sodium hypochlorite solution for 1 min, rinsed twice in water, and then inoculated with AH1 using a sprayer, while leaves treated with sterile water served as negative controls.The experiment was conducted four times, and each repeat contained 10 plants. Pathogenicity testswereperformedinthe greenhouseat25°C with a 12 h photoperiod. Partial yellow lesions were observed 3 days of post-inoculation. As the disease progressed, the tawny color gradually spread across the leaf and the tip became dark brown within 7 days. The necrosis expanded and some small leaves were completely affected within 2 weeks. The pathogen was re-isolated from the lesions and re-identified through morphological traits and sequence analysis. A. alternata have been reported to cause leaf diseases in a variety of cereal crops, vegetables, and fruits across China (Sun et al. 2021; Zheng et al. 2015), which cause significant crop loss. To our knowledge, this is the first report of A. alternata causing leaf blight of H. coronarium in the world. More surveys are needed to explore the epidemiology and management strategies for disease caused by A. alternata in Southwest China.

Chemical differences between brown centre and white macadamia kernels

Nuts are susceptible to many quality issues, including discoloration defects in kernels. These discolouration defects can affect their market value and result in significant economic losses to both growers and processors. In macadamia, this quality issue is called brown centres and has been related to storage at high temperatures and moisture concentrations. However, the chemical differences between brown centre and white macadamia kernels have not previously been studied. We aimed to determine the chemical differences between macadamia kernels with brown centre defects and kernels that were white and cream coloured. We used brown and white kernel samples from 20 commercially processed batches of macadamia. From each batch, we selected brown centre samples and compared their chemistry with white kernel samples from the same batch. We compared the moisture concentration, sugar concentration, nutrient concentration, fatty acid profile and peroxide value of brown centre defect and white kernel samples. Our results showed that brown centres had a higher moisture concentration compared with white kernel samples (5.16 % vs 2.91 %, respectively, P < 0.05). Reducing sugars of fructose (0.61 %) and glucose (0.53 %) concentrations were only detected in brown centre, although the sucrose concentration was not significantly different. Most nutrient concentrations were higher in brown centres compared with white kernels. Nitrogen concentration was significantly higher in brown centre than white centres (2.10 % vs 1.82 %, respectively, P < 0.05). Calcium concentration was also higher in brown centre than white macadamia kernels (608.44 mg/kg vs 419 mg/kg, respectively, P < 0.05). Our study suggests that high moisture concentrations in macadamia kernels could have triggered the hydrolysis of sucrose into reducing sugars (fructose and glucose). In addition, reducing sugars and proteins can react in the Maillard reaction to form brown colouration in nuts. This is the first study to comprehensively compare the nutritional composition of brown centres and white macadamia kernels. We recommend appropriate drying and storing of macadamia nuts to prevent sucrose degradation into reducing sugars and decrease the formation of brown centres through the Maillard reaction.

Molecular and morphological studies reveal a new species of Panaeolus (Agaricales, Basidiomycota) from Punjab, Pakistan

Panaeolus punjabensis M. Asif, Q. Firdous, A. Izhar, Niazi &amp; Khalid sp. nov. was collected from three different localities (Bahawalnagar, Kasur, and Lahore) in Punjab, Pakistan. Morphological observations and phylogenetic analyses based on nuclear encoded internal transcribed spacers (ITS1-5.8S-ITS2 = ITS) and D1/D2 domain of large subunit (28S) rDNA confirmed the taxonomic distinctness of this species. The new species is potentially hallucinogenic and characterized by a parabolic pileus with a light brown center, broadly fusiform basidiospores, presence of cheilocystidia, pileocystidia, and caulocystidia, and absence of pleurocystidia and clamp connections. The DNA sequences of the species clustered together in a well-supported distinct clade. We present a detailed description, photographs, and line drawings, and elucidate and discuss the phylogenetic position of the new species. Morphological comparisons with phylogenetically and morphologically allied species are discussed.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

First report of leaf spot on passion fruit in China, caused by Alternaria alternata.

Passion fruit (Passiflora edulis Sims), which is native to South America, is an important fruit crop in tropical and subtropical countries. Passion fruit growing areas have increased rapidly in southern China. In 2018 to 2019, circular spots on passion fruit were observed in Shangsi, Guangxi, China (21°15'N, 107°98'E). The disease occurred from June to April of the following year. The disease incidence was generally between 10% to 30%, but could reach up to 50% in purple passion fruit 'Tainong No.1'. The initial lesions on the fruits were small, with a brown center and a greasy margin, and then became sunken and lighter brown with a diameter of about 1 cm in later stages. The spots on the leaves were often surrounded by a yellow halo and turned into larger lesions after coalescence.. Five typical symptomatic fruit and leaves were collected from Shangsi county for the presumed pathogen isolation. Section of the samples were surface sterilized to isolate the fungus on potato dextrose agar (PDA) at 28°C. Five fungal isolates with similar morphology on PDA were obtained by single spore isolation. Colonies at the age of 7 days accompany with flourishing aerial hyphae, showed surface color varying from white to grey. Conidia were ovate or elliptic, light brown to brown, with 2 to 5 diaphragms, 0 to 4 longitudinal-oblique diaphragms, and mostly 8.2 to 36.7 μm × 5.4 to 15.8 μm. The morphology of the fungus resembled Alternaria alternata (Fr.) Keissl (Simmons, 2007). Each of the five isolates (SF-001, SF-002, SF-003, SF-004 and SF-005) was molecularly identified using genomic regions of 18S nrDNA (SSU), 28S nrDNA (LSU), RNA polymerase second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) (Jayawardena et al., 2019). Nucleotide sequences of SSU (MZ275254, ON055696, ON055697, ON055698 and ON055699), LSU (MZ275253, ON062947, ON062948, ON062949, ON062950), RPB2 (MZ275251, ON055377, ON055378, ON055379 and ON055380), ITS (MW866522, MW866523, ON053451, ON053452 and ON053453), GAPDH (MZ286628, ON055381, ON055382, ON055383 and ON055384) and TEF1 (MZ275255, ON055373, ON055374, ON055375 and ON055376) were deposited in GenBank database. The LSU, GAPDH and TEF1 sequences showed 100% identity with A. alternata in NCBI (KX609773, MK683852 and MK637432, respectively). The SSU, RPB2 and ITS sequences showed 99% identity to A. alternata (U05194, MK605898 and MN856409, respectively). In pathogenicity test (Zhang et al., 2020), 3-month-old grafted 'Tainong No.1' seedlings and mature fruit were used. Five-mm-diameter mycelial plugs taken from 7-day-old PDA colonies of each of 5 isolates were placed on the leaves and fruit that were wounded with a sterilized needle to form 3 pinpricks. Sterile PDA plugs were used as control. Three plants and three fruits were used in each treatment, and the test was repeated twice. The inoculated plants and fruit were kept in plastic bags and grown in a chamber at 28℃. Typical lesions were observed on inoculated plants and fruit after 3 days, but the controls remained healthy. A. alternata was consistently reisolated from these typical lesions. Previously, leaf spot on passion fruit caused by A. alternata has only been recorded in New Zealand (Rheinländer, 2010). To our knowledge, this is the first report of A. alternata (Fr.) Keissl. causing leaf spot on passion fruit in China. The identification of the pathogen may help to take effective management strategies of controlling this disease.

First report of leaf spot disease caused by Xenodidymella glycyrrhizicola on Glycyrrhiza glabra

Glycyrrhiza glabra (liquorice) is a perennial shrub belonging to the Fabaceae family. It is a native of Mediterranean areas and primarily cultivated for its extracts which are used in the pharmaceutical and food industries, and in the manufacture of food supplements (Hayashi & Sudo, 2009). Several foliar diseases, such as powdery mildew, rust and leaf spots, have been reported to endanger the long-term viability and production of liquorice (Farr & Rossman, 2023). In 2022, a leaf spot disease was observed on c. 40% of the two-year-old plants in a liquorice field in Ankara, Turkey. Leaf spots were brown-to-black in colour, sometimes with a light brown centre. Lesions coalesced to large areas of blighted tissue with age (Figure 1a). Branches and petioles also became covered in oval lesions (Figure 1b). Pieces (5 × 5 mm) from lesion margins were surface sterilised in 70% ethanol (30 seconds) and 1% NaOCl (1 minute) and rinsed three times with sterile water. Phoma-like colonies were isolated from surface-sterilised parts of diseased tissues on potato dextrose agar (PDA). One representative isolate (Xg_Gg01), purified using the hyphal tip method, was selected for molecular, morphological and pathogenic characterisation and transferred to plates containing fresh PDA, oatmeal agar (OA), and malt extract agar (MEA) for seven days at 25°C with 12 hr light and 12 hr dark cycles. Colonies on MEA reached about 45 mm in diameter, margin regular and white, brown towards the centre, which was brown and orange due to conidial discharge from pycnidia (Figure 2a). Colonies reaching about 50 mm in diameter on the OA were marginally regular and olivaceous, with a colour change to black due to pycnidia formed towards the centre (Figure 2b). Colonies on the PDA reached about 43 mm in diameter, and the margin was regular and pale olive; the middle and centre were black due to dense pycnidia, and light brown conidial discharge was observed in the centre (Figure 2c). Pycnidia were mostly superficial or immersed in agar, ostiolate, 230–470 × 190–365 μm, and globose or subglobose and brown, becoming irregular-shaped and black with age (Figure 3a). Conidia were aseptate, variable in size and shape, oblong, broadly ellipsoidal, hyaline, becoming pale brown with age and measured 6.1 ± 9.3 × 3.4 ± 6.4 μm (Figure 3b). The internal transcribed spacer (ITS) of ribosomal DNA, partial 𝛽-tubulin gene (TUB2) and RNA polymerase second largest subunit (RPB2) loci of isolate Xg_Gg01 were amplified and bidirectionally sequenced with the universal primer sets; ITS1/ITS4 (White et al., 1990), BT2a/ BT2b (Glass & Donaldson, 1995), and 5f2 (Reeb et al., 2004)/7cr (Liu et al., 1999), respectively. A BLAST search of the resultant sequences (GenBank Accession Nos. OQ568230 for ITS, OQ571627 for TUB2, and OQ571628 for RPB2) showed 99.61–100% nucleotide identity with the holotype strain CBS 684.97 of X. glycyrrhizicola from New Zealand. The combined maximum-likelihood tree of ITS, TUB2, and RPB2 regions with 1000 bootstrap replicates showed that the isolate from the current study was clustered with strains of X. glycyrrhizicola derived from GenBank with strong support (Figure 4). Koch's postulates were fulfilled by spraying 10 ml of a spore suspension (106 spores/ml prepared in a 0.1% Tween-20 solution) of isolate Xg_Gg01 on each of five two-month G. glycyrrhizicola plants in trial pots. The plants were covered with plastic bags for 24 hr post inoculation. After seven days, spots were observed on the inoculated plants’ leaves and petioles (Figure 1c). No symptoms were observed on control plants, sprayed with sterile distilled water only. A morphologically identical X. glycyrrhizicola was isolated from affected tissues. To the best of our knowledge, this is the first report of X. glycyrrhizicola associated with leaf spots on G. glabra (Farr & Rossman, 2023). The fungus was previously reported on Glycyrrhiza lepidota in Iran (Ahmadpour et al., 2022) and New Zealand (Hou et al., 2020). However, G. glabra constitutes a new host record for this pathogen in Turkey and worldwide.

First report of Coniella vitis causing white rot on Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) in China.

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) belongs to the genus of Parthenocissus and Vitaceae family, which is very common in vineyards and where wild grape occurs (Bergh et al., 2011). In September of 2021, a severe white rot disease was observed on Virginia creeper around the vineyard of wine grapevine (Cabernet Sauvignon) located in Penglai city (37º 75'38" N, 120º 84'28" E), Shandong province of China. The disease incidence was about 75%, and infected leaf of Virginia creeper exhibited irregular necrotic lesion with brown center, and most lesion occurred on leaf margin, black pycnidia were also observed on the infected leaf at the late stage of infection. To determine the causal agent, symptomatic leaves with typical lesions were cut into small pieces (5 mm × 3 mm), surface sterilized with 75% ethanol for 1 min, followed by three times rinsed in sterile water. Leaf sections were plated onto potato dextrose agar (PDA) medium and incubated at 28°C for 3 days. Totally, five isolates (referred to as JD01, JD07, JD09, JD12 and JD16) were collected and transferred on to fresh PDA medium for incubation at 28°C. Seven days later, colonies on PDA plates had crenulated edges with concentric rings, the upper surface of colonies was mostly flat and white with many pycnidia. The conidia were hyaline at immature and became brown later, spherical or ellipsoid, aseptate, and 7.92 ± 1.20 μm × 5.18 ± 0.61 μm (n=50), length : width ratio is nearly 2. Morphologically, the isolates were identified as Coniella vitis (Chethana et al., 2017). Further to confirm the fungal species, the internal transcribed spacer region (ITS) of the ribosomal RNA gene, large subunit rRNA gene (LSU) and the translation elongation factor 1-alpaha gene (TEF1-α) were amplified using primers ITS1/ ITS4, LR7/ LROR, and TEF1- 728F/ TEF1- 986R (Chethana et al., 2017; Raudabaugh et al., 2018). The amplification products were sequenced and deposited in GenBank database. The sequences were compared to type sequences in GenBank. The results showed that ITS (GenBank accession numbers ON329769, ON329770, ON329771, ON329772 and ON329773), LSU (ON358423，ON358424, ON358425, ON358426 and ON358427) and TEF (ON297671, ON229071, ON229072, ON229073 and ON297672) sequences of the five isolates were 99.66%, 96.90% and 98.79% identical with the sequences data from C. vitis isolates in GeneBank (MFLUCC 18-0093, JZB3700020 and MFLUCC 18-0093, respectively). Furthermore, concatenated sequences of the three genes (ITS, LSU and TEF) were used to conduct a phylogenetic tree using maximum likehood MEGA-X (Raudabaugh et al., 2018). The phylogenetic analysis showed that the five isolates (JD01, JD07, JD09, JD12 and JD16) belong to C. vitis clade among the 41strains of Coniella spp. In the pathogenicity tests, detached leaves of Virginia creeper (1-year-old) were inoculated with mycelia plugs (5 mm diameter) (form 3-day-old of isolate JD07 culture), and control were inoculated with PDA plugs (5 mm diameter). Virginia creeper live plants (1-year-old) were inoculated with conidial suspension (2.5×106 spores/ml) of the isolate JD07 of one week old, and control plants were inoculated with sterile water. All treated Virginia creeper plants (detached leaves) were placed in a greenhouse maintained at 28°C and 95% relative humidity. Virginia creeper plants (detached leaves) inoculated with the conidial suspension (fungal mycelia) had brown lesion on leaves, the disease symptoms were similar to those observed in field. No such symptoms were observed on control plants (detached leaves). The pathogen was reisolated from inoculated Virginia creeper plants and re-identified, thus fulfilling Koch's postulates. C. vitis had been reported to cause grape white rot in China (Chethana et al., 2017). Virginia creeper, as an excellent host of C. vitis, will increase the transmission risk of the pathogens. To our knowledge, this is the first report of C. vitis causing white rot on Virginia creeper, and this finding will provide useful information for developing effective control strategies for white rot disease.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

First report of Fusarium falciforme causing basal rot of onion (Allium cepa) in India.

Onion (Allium cepa, L) is a very important vegetable crop in India. India is the second largest producer of onion in the world and the crop is grown on more than 1.22 million hectares. Fusarium Basal Rot (FBR) is an economically important diseasef onion that causes considerable losses in onion production up to 50% in field and 30-40% during post-harvest storage of bulbs (Gupta and Gupta 2013; Rajamohan et al. 2019). Onion plants showing chlorosis, twisting, wilting, necrosis, bulb discoloration, rot in the basal parts of bulb and roots typical to FBR were observed, in a field trial of 36 onion cultivars during October 2020 in Bangalore, Karnataka. FBR incidence varied from 30-100% in this field (Fig 1 a-d). Symptomatic bulbs were washed with water, basal plate and fleshy leaves cut into 0.5 to 1 cm-size, surface disinfected with 1.5% sodium hypochlorite (NaOCl) for 3 min, and rinsed with sterile distilled water. Twenty pieces were placed on potato dextrose agar (PDA) in Petri plates and incubated at 25°C for 7 days. Colonies from single-spore isolates on PDA showed abundant white aerial mycelium. Colonies showed light pink or purple coloration on the reverse side of the culture plate with brown center (Fig 1e-f). Macroconidia were 19.13 to 28.35 (mean= 24.2) × 4.29 to 6.06 (mean= 5.05) µm, hyaline, falcate, with slightly curved apexes, and three to five septa. Microconidia were cylindrical to ellipsoid, aseptate, hyaline 8.20 to 12 (mean=10.0) × 3.55 to 4.79 (mean= 4.29) µm (Fig 1g). Chlamydospores were round, intercalary, hyaline, single or in chains (Fig 1h). Two isolates (IBFF-09 & IBFF-10) were analyzed for internal transcribed spacer-ITS (White et al. 1990) and translation elongation factor 1-α (tef1) gene (O'Donnell et al. 1998) by polymerase chain reaction (PCR) and sequenced. ITS and partial tef1 gene sequences of isolates IBFF-09 and IBFF-10 were submitted to the NCBI database (GenBank accession # ON394614&ON026859; # ON409480, ON093166 respectively). Phylogenetic analysis of tef1 gene placed the isolates with F. falciforme (Fig 1i). A pathogenicity test was performed by dipping roots of 28 days old healthy onion seedlings of a susceptible genotype 16/7Y GR3 into a conidial suspension (1 × 104 conidia/ml) of isolate IBFF-10 for 15 min and then transplanting the plants into pots containing sterilized potting mix. Inoculated plants developed typical symptoms of FBR and were all dead by 20 days post inoculation (Fig 1j) while the non-inoculated controls remained healthy. Pathogen was re-isolated from infected plants and showed the same morphology, ITS and tef1 sequence similarity as the original isolate, thus fulfilling Koch's postulates. Basal rot of onion by F. falciforme is reported from Mexico (Tirado-Ramírez et al. 2021). Till date, only F .oxysporum, F. proliferatum and F. solani have been implicated in onion FBR in India (Lee et al. 2021; Rathore and Patil, 2019). F. falciforme however, i prevalent in India and is reported to infect other crops (Gangaraj et al. 2022; Gupta et al. 2019; Homa et al. 2018). There is a high probability that this pathogen is contributing significantly to basal rot disease but it has not been reported yet. To our knowledge, this is the first report of F. falciforme infecting onions in India. In order to develop FBR resistant onion cultivars it is critical to identify and study the response of onion genotypes to different Fusarium spp causing the disease.

Sampling and Categorization of Households for Research in Urban India

Conventional sampling methodologies for citizens/households in urban research in India are constrained due to the lack of readily available, reliable sampling frames. Voter lists, for example, are riddled with errors and, as such may not be able to provide a robust sampling frame from which a representative sample can be drawn. The Jana–Brown Citizenship Index project consortium (Janaagraha, India; Brown University, USA) has conceptualized a unique research design that provides an alternative way on how to identify, categorize and sample households (and citizens within) in a city in a representative and meaningful way. The consortium consists of the Janaagraha Centre for Citizenship and Democracy, based in India, and the Brown Center for Contemporary South Asia, part of Brown University, USA. The methodology was designed to enable systematic data collection from citizens and households on aspects of citizenship, infrastructure and service delivery across different demographic sections of society. The article describes how (a) data on communities that are in the minority, such as Muslims, scheduled castes (SC) and scheduled tribes (ST), were used to categorize Polling Parts to allow for stratified random sampling using these strata, (b) geospatial tools such as QGIS and Google Earth were used to create base maps aligning to the established Polling Part unit, (c) the resulting maps were used to create listings of buildings, (d) how housing type categorizations were created (based on the structure/construction material/amenities, etc.) and comprised part of the building listing process, and (e) how the listings were used for sampling and to create population weights where necessary. This article describes these methodological approaches in the context of the project while highlighting advantages and challenges in application to urban research in India more generally.

Screening Table Beet and Swiss Chard for Resistance to Pseudomonas syringae pathovar aptata

Bacterial leaf spot (BLS) has emerged in the last few decades as an economically important disease of both table beet (Beta vulgaris ssp. vulgaris) and Swiss chard (Beta vulgaris ssp. cicla). BLS is caused by Pseudomonas syringae pv. aptata, which is spread readily on infected seeds. Symptoms appear as circular to irregular shaped, with a tan to dark brown center and a very dark border. Disease incidence and severity is dependent on cool, humid conditions and can vary widely year to year depending on the environment. Both the vegetative and reproductive phases of these biennial crops are susceptible to the pathogen. Table beet and Swiss chard commercial cultivars (n = 21), table beet breeding lines (n = 5), and table beet plant introductions (PIs) (n = 26) were screened for response to spray inoculation with P. syringae pv. aptata in a controlled greenhouse setting. Plants were rated for severity of symptoms using percent of the area of each pair of leaves (leaf set) with symptoms and an overall plant score assigned based on the scores for each leaf pair. Accessions varied in BLS susceptibility. PI accessions were most variable, with the area under the disease progress curve (AUDPC) ranging from 1.33 to 8.75. Highly significant differences among PIs were detected for disease scores in the vegetative stage, beginning 21 days after inoculation. Screens during the reproductive growth stage showed the least variation in AUDPC among PIs. Although cultivars varied less than PIs, good BLS resistance (low disease scores) was noted for ‘Touchstone Gold’, ‘Kestrel’, ‘Bull’s Blood’, ‘Rainbow’ chard, as well as PIs 222234 and NSL 28026. Accessions W451C, Red Cloud, Detroit Dark Red, and NSL 28020 were highly susceptible. There was no consistent association between disease score in the vegetative and reproductive phases, suggesting that breeders may need to screen for BLS in both phases of the biennial life cycle. The more resistant PIs or cultivars identified in this study can be used in future efforts to breed for host resistance to BLS and to establish mapping populations to better understand the genetic control of resistance, to aid in breeding efforts.