16-hour Photoperiod Research Articles

In vitro culture of Campomanesia pubescens under different light qualities: a morphoanatomical and physiological characterization

Campomanesia pubescens (gabiroba) is a fruit tree that is native to the Cerrado and that has commercial potential and medicinal properties. It has recalcitrant seeds that do not tolerate desiccation and storage. Here, C. pubescens plants were grown in vitro under different light qualities. Light-emitting diodes (LEDs) were used at wavelengths for white (W) and blue in combination with red in the proportions BR (1:1), BR (1:3) and BR (3:1), at 100 ± 5 μmol m-2 s-1 over a 16-hour photoperiod. The leaf anatomy, chlorophyll a fluorescence, chloroplast pigments and malondialdehyde (MDA) content were evaluated. Studies on Cerrado plants grown in vitro focusing on light quality and its effects on plant development and growth are scarce. Records of anatomical and physiological C. pubescens characteristics when grown in vitro under different lighting qualities using LEDs are nonexistent. In this context, the aim of this study was to analyze the anatomical and physiological responses of C. pubescens seedlings from in vitro cultivation under different light qualities, to support future studies on the propagation and conservation of this species. This work is a pioneer for this species. When the plants were cultivated in combinations of blue/red LEDs 1:1 and 3:1, they yielded higher biomass, presenting higher epidermis and chlorenchyma values with greater stomatal density and functionality, better photosynthetic efficiency, higher Y(II) values, qP and higher total chlorophyll concentrations, and thus these lights did not cause oxidative damage compared to the white (control) LEDs. This study contributed to a better understanding of the anatomical and physiological changes in C. pubescens plants grown in vitro under different light qualities. C. pubescens plants exhibited varied anatomical and physiological characteristics depending on the spectrum of light used here. The combinations of BR (1:1) or BR (3:1) LEDs are promising for the propagation of the species, given their positive influence on most of the studied traits.

In vitro mass propagation of Dendrocalamus asper (Giant bamboo) through direct organogenesis

Dendrocalamus asper (giant bamboo) is a clumping type of bamboo belonging to the Poaceae family. Due to its economic and environmental value, demand for this species has increased tremendously. Conventional propagation methods have limitations due to low seed viability and the lack of healthy clumps. Therefore, an in vitro mass propagation protocol was developed to provide healthy plants for large-scale plantations. Seeds were used as the explant and they were surface sterilized and cultured on MS medium free of growth regulators. Nodal segments taken from in vitro germinated seedlings were used for shoot initiation. The best medium for shoot induction (MS medium supplemented with 0.0–2.5 mg/L BAP), best medium for multiple shoot induction (MS medium supplemented with 0.0–5.0 mg/L BAP), effect of shoot cluster size (shoot clusters containing 1–4 shoots) and effect of physical state of the medium (semisolid and liquid media) on multiple shoot induction were determined using shoots per node, mean shoot length and mean number of leaves per shoot after 6 weeks of incubation. Elongated shoots were transferred into a rooting medium and the best medium for root induction (MS medium supplemented with 0.0–5.0 mg/L IBA and IAA) and effect of cluster size (shoot clusters containing 1–4 shoots) on rooting were determined using the number of roots and root length after 6 weeks of incubation. All the cultures were maintained under a 16-hour photoperiod. Well-developed plantlets were transferred to coir pellets and after four weeks transferred into different potting mixtures containing different combinations of sand, compost and coir dust. Unless otherwise mentioned there were at least twenty replicates in all treatments. The highest mean number of shoots per node (16.87±0.52), mean shoot length (4.12±0.27 cm) and mean number of leaves per shoot (4.80±0.33) were observed in the MS medium supplemented with 1.0 mg/L BAP. The MS medium supplemented with 2.0 mg/L BAP was the best for multiple shoot induction, shoot cluster with 3 shoots was the best cluster size and the liquid medium had a better effect on shoot multiplication. The half-strength MS medium supplemented with 2.0 mg/L IBA was the best for in vitro root induction with the highest mean number of roots (7.15±0.77) and a mean root length of 10.79±1.11 cm. Shoot clusters with 3 shoots was the best cluster size for root induction. A sand: compost: coir dust (1:1:1) mixture was the best potting mixture giving 100% survival. These findings provide a reliable micropropagation protocol for D. asper, which holds great promise for meeting the growing demand for bamboo resources and promoting sustainable bamboo cultivation.

First report of soybean root rot caused by Fusarium proliferatum in the Republic of Korea.

Fusarium species are widespread soilborne pathogens that can cause damping-off, root rot, and wilting in soybean [Glycine max (L.) Merrill], subsequently leading to significant yield suppression. Several Fusarium spp. have already been documented for their pathogenicity on soybean plants in the Republic of Korea. The nationwide monitoring of soybean diseases continues to identify new pathogenic Fusarium spp. In 2016, five plant samples at R3-R4 growth stages, showing symptoms of wilting in the upper parts and root rot, were collected in Suwon, Gyeonggi, Republic of Korea. Fungal colonies were obtained from the diseased root samples, with the surface sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and placed on water agar at 25°C. Five isolates were collected and purified by single-spore isolation. The fungal mycelium was subsequently cultivated on potato dextrose agar for ten days. The isolates produced abundant, aerial, and white mycelium and became purple in old cultures. Macroconidia were slender, falcate to almost straight, usually 3 to 5 septated, and thin-walled. Microconidia were formed in chains from polyphalides, clavate or oval, usually single-celled with a flattened base. These characteristics of isolates were consistent with the description of F. proliferatum (Leslie and Summerrell 2006), and the representative isolate 16-19 was selected for molecular identification to confirm its identity as F. proliferatum. Two evolutionarily conserved genes, the translation elongation factor 1-alpha (EF-1α) and the second-largest subunit of RNA polymerase II (RPB2) genes, were partially amplified using the primers described by O'Donnell et al. (2008), resulting in nucleotide sequences of 680 and 382 base pairs, respectively. These two sequences (GenBank accession numbers: OQ992720 and OR060666) showed 100 and 99.5% identity to the EF-1α and RPB2 of F. proliferatum A40 (GenBank accession numbers: KP964907 and KP964842). For the Petri-dish pathogenicity assay (Broders et al. 2007), five surface-sterilized seeds were placed on water agar media with either sterile water or actively growing '16-19' culture. After 7 days of incubation in a growth chamber (25°C; 12-hour photoperiod), brown lesions were observed on the roots of the inoculated plants, while no symptoms were observed in the sterile water-treated controls. The experiment was conducted three times. For root-cut pathogenicity assay, conidial suspension (1×106 conidia/ml) of the isolate '16-19' was prepared with harvested mycelia cultured on PDA for 10 days with sterile water. The roots of 10-day-old soybean seedlings were partially cut and soaked in either the suspension or sterile water for 2 hours. The seedlings were transplanted into 12 cm plastic pots (11 cm in height) and grew in a greenhouse (26 ± 3°C, 13-h photoperiod). The experiment followed a completely randomized design with three replicates (i.e. three plants in a pot), and it was repeated twice. The inoculated plants began to wilt 7 days after inoculation, while the sterile water-treated controls remained healthy. Ten days after inoculation, all plants were collected, washed under running tap water, and evaluated for the presence and severity of root rot using a 0-4 scale (Chang et al. 2015). The inoculated plants exhibited reduced vigor and developed dark brown lesions on their roots. F. proliferatum was reisolated from symptomatic root tissues of the infected plants, while not from those of the controls. Its colony and spores were morphologically identical to those of the original isolate. F. proliferatum was previously reported as a causative agent of soybean root rot in the United States (Díaz Arias et al. 2011) and Canada (Chang et al. 2015). This is the first report of soybean root rot caused by F. proliferatum in the Republic of Korea. This finding implies that F. proliferatum may potentially threaten soybean production in the Republic of Korea and suggests that effective disease management strategies should be established for soybean protection against the disease, along with continuous surveillance.

Dark brooder pullets are less active than Non-brooder pullets, but show similar level of synchronisation at the resource areas

Maternal care in commercial chickens can partially be replaced with dark brooders that offer heat and a dark area to rest and possibly avoid negative social interactions. Previous research has established the positive effects of dark brooders on reducing disturbance during resting in young pullets and injurious pecking in pullets and laying hens, which could reduce overall activity levels. The objective of this study was to employ precision livestock farming techniques to compare the overall activity levels and behavioural synchrony at resources in pullets reared with dark brooders until 41 days of age and those reared with whole house heating. Four brooder treatments, varying in size (Small/Large) and deployment method (Raised/Fixed), which could have implications for commercial use, were employed. Activity of the whole pen floor area and behavioural synchrony in drinker and feeder areas were automatically assessed over a 12-hour photoperiod at 10 and 60 days of age. Pixel change detection gauged overall activity across the pen, while an object detection model evaluated pullet behavioural synchrony. The analysis revealed increased activity levels in non-Brooder pullets compared to those in most Brooder treatments at both 10 and 60 days of age. However, no significant differences in behavioural synchrony were observed between Brooder and non-Brooder pullets. The underlying mechanism of dark brooder’s effects on the activity levels and behaviour synchrony remains unclear, but the observed reduction in activity levels in brooded pullets likely corresponds to increased resting behaviour and reduced injurious pecking. Furthermore, it is noteworthy that only a small percentage of pullets (up to 57.14%) were observed to use the resources simultaneously. This underscores the importance of conducting additional research to delve into the effects of resource allocation on both behavioural synchrony and activity levels in pullets. We observed minimal differences in the behaviour of pullets raised under different brooder types, suggesting that the simplest (Small-Fixed) brooders may be suitable for commercial use.

Improve Cannabis sativa micropropagation through increasing air change rate in photoautotrophic and traditional tissue culture

Micropropagation is essential for pathogen-free plant materials. However, the conventional in-vitro micropropagation approach poses challenges for optimal growth and development. This study enhances Cannabis sativa micropropagation by implementing a photoautotrophic tissue culture with increased air change rates. This method relies exclusively on photosynthesis for carbohydrate synthesis, eliminating the need for exogenous sugar supplementation. To overcome the limitations of conventional in vitro culture, the photoautotrophic culture requires higher air exchange in the vessel to supply CO2 for photosynthesis. The research evaluates the effects of varying air change rates, comparing conventional tissue culture (TC) with photoautotrophic micropropagation (PAM). Nodal explants, each with 3–4 fully expanded leaves, were transferred to PAM or TC media in Magenta boxes, undergoing four distinct air change rate treatments: 0.5 (control), 4.1, 7.9, and 14.5 air change per hour. Magenta boxes were placed under controlled chamber conditions, including a photosynthetic photon flux density of 100 µmol·m−2·s−1, 18-hour photoperiod, 25 °C temperature, 70 % relative humidity, and CO2 concentration of 553 µmol·mol−1; data were collected after a 28-day period. Increasing the air change rate in TC (from 0.5 to 7.9 h−1) and PAM (from 0.5 to 14.5 h−1) resulted in a significant 25.1 % and 146.6 % increase in shoot dry mass, respectively. The number of axillary buds per plant exhibited a linear increase response to increasing air change rates only under PAM; as well as a significant difference between PAM (11.5) and TC (8.3) under an air change rate of 7.9 h−1. Particularly advantageous for PAM was simultaneous root growth, escalating from 5.2 to 44.3 mg as the air change rate rose from 0.5 to 14.5 h−1. Implementing the photoautotrophic culture practice not only accelerated Cannabis micropropagation but also maintained outcomes comparable to traditional sugar-based tissue culture, contributing to cost-effective cultivation practices in the Cannabis and tissue culture industries.

First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

First Report of Anthracnose Leaf Spot Caused by Colletotrichum americae-borealis on Potentilla anserina in China.

Potentilla anserina L. has an abundance of bioactive compounds and is widely recognized for its diverse applications in traditional medicine and as a food. In August 2023, typical symptoms of anthracnose were observed in 80% of P. anserina plants in Harbin, China. Symptoms, characterized by reddish-brown spots, tend to occur more frequently on leaves closer to the ground. They initially appeared as oval or irregular circles, measuring 1 to 3 mm in diameter, and later merged into larger patches surrounded by chlorotic areas on the leaves. Twenty leaves exhibiting characteristic symptoms were sampled. Each leaf was sectioned into 5×5 mm pieces at the interface between the diseased and healthy tissues. The sections were disinfected sequentially with 75% ethanol for 30 s, followed by 1% NaClO for 2 min, rinsed three times in sterilized distilled water. Post air-drying, samples were cultured on potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d, yielding nine morphologically similar single-spore isolates (JTC1 to JTC9). The colonies initially displayed gray aerial mycelia, becoming pale brown, accompanied by numerous black microsclerotia. The acervuli appeared black, protruded from the surface of the medium, and were adorned with dark brown setae. Setae (n=50) ranged from 58.4 to 188.2 μm in length, appearing dark brown to black, with smooth walls, rounded tips, swollen bases, and containing 1 to 4 septa. The conidia were hyaline, aseptate, cylindrical to spindle-shaped, with blunt and rounded ends, measuring 13.7 to 18.3 μm in length and 3.4 to 4.3 μm in width (n=50). Morphological analysis indicated a close affinity with Colletotrichum americae-borealis (Damm et al. 2014). For molecular identification, genomic DNA was extracted from three representative isolates (JTC1, JTC2, and JTC3).The ITS, HIS3，GAPDH, and ACT genes were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences were submitted to GenBank (ITS: PP338190 to PP338192; HIS3: PP355770 to PP355772; GAPDH: PP355773 to PP355775; ACT: PP355776 to PP355778). BLAST analysis showed 99 to 100% identity with C. americae-borealis type strain CBS 136232 (GenBank accessions: KM105224, KM105364, KM105579, and, KM105434, respectively). Multigene phylogenetic analysis positioned the three isolates close to C. americae-borealis. Pathogenicity tests were performed twice on 6-week-old P. anserina seedlings (cv. Qinghai Juema 1) in a greenhouse. A conidial suspension of the JTC1 isolate (1×10⁵ conidia/ml) was sprayed applied to ten pots, each containing two seedlings, and the plants in the control pots were sprayed with sterile distilled water. Two weeks after inoculation under greenhouse conditions (26/22°C day/night temperature, 12-hour photoperiod, 90% relative humidity), the inoculated seedlings exhibited brown spots and necrotic lesions similar to those observed in the field, C. americae-borealis was successfully reisolated from these symptomatic tissues. To the best of our knowledge, this is the first report of C. americae-borealis causing leaf spot on P. anserina in China. Anthracnose caused by C. americae-borealis is associated with leaf spot disease in oats (Wang et al. 2022), alfalfa (Li et al. 2021), and licorice (Lyu et al.2020). However, C. americae-borealis poses a significant threat to P. anserina in China as well, highlighting the urgent need to develop effective disease management strategies.

Evaluating Organic Fertilizers and Microbial Inoculation for Soilless and Hydroponic Crop Production

Because hydroponic operations in the United States can be certified as organic, and because the price of chemical fertilizers has increased, there is an increasing interest in using organic fertilizers and beneficial microorganisms for controlled-environment agriculture. However, there is a scarcity of information regarding their effectiveness and application methodologies. We investigated the effects of inoculating Azospirillum brasilense and Rhizophagus intraradices and using organic fertilizers on growing lettuce (Lactuca sativa ‘Cherokee’) and tomato (Solanum lycopersicum ‘Red Robin’) young plants in an indoor vertical farm. Seeds were sown in rockwool substrate, with A. brasilense (1.05 × 108 colony-forming units⋅L−1) or R. intraradices (580 propagules⋅L−1) applied weekly via subirrigation. Seedlings received chemical fertilizer, organic fertilizer derived from corn steep liquor and fermented fish by-products, and food waste-derived organic fertilizer at 100 ppm total nitrogen every 2 or 3 days. They were grown indoors at 23 °C under light-emitting diode lighting at a photosynthetic photon flux density of 200 μmol⋅m−2⋅s−1 with an 18-hour photoperiod. Lettuce under organic fertilizers had 75% lower shoot fresh mass and 64% less shoot dry mass compared with lettuce under chemical fertilizer. Similarly, tomato seedlings with organic fertilizers had fewer leaves, 75% less shoot fresh mass, and 67% less shoot dry mass. In both lettuce and tomato, the macronutrient and micronutrient concentrations in plant tissues were generally similar regardless of fertilizer treatments, but nitrogen use efficiency and nitrogen uptake efficiency were lower under organic fertilizers compared with those under chemical fertilizer. The inoculation of A. brasilense or R. intraradices showed limited effects on plant nutrient uptake, nutrient concentrations, and seedling growth in both lettuce and tomato. Further research is necessary to optimize application methods for organic fertilizers and beneficial microorganisms to fully harness the benefits of sustainable alternative fertilizers in soilless and hydroponic crop production.

First Report of Collar and Root Rot of Lettuce Caused by Plectosphaerella cucumerina in Serbia.

In March 2021, unusual plant stuning, collar, and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage was observed in two commercial fields in Serbia (44°58'N, 20°32'E; 44°45'N, 20°43'E). Disease incidence in the fields (≈ 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar, wet root rot, and lesions becoming necrotic. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields, and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar (PDA). Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, gradually becoming salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1×1.2 to 3.7 μm (n = 100)). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O'Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS: OR880564 and D1/D2: OR880567). Sequence analysis revealed 100% nucleotide identity with P. cucumerina isolates from different countries deposited in the NCBI GenBank, including isolate MH860704 (Vu et al. 2019) (ITS region) and isolate KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on the combined ITS and D1/D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). The combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Palm et al., 1995; Carlucci et al., 2012). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1×105 conidia/ml). The 10 inoculated plants were transplanted into 1 L pots containing sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-hour photoperiod (Cai et al., 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch's postulates were completed by confirming the identity of reisolates based on morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crop in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, the Netherlands, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.

Microtuber Induction and Plant Regeneration of Potato (Solanum tuberosum L.): A Review

Potato (Solanum tuberosum L.) is one of the most economically significant annual vegetable crops occupying the fourth position worldwide among all the food crops. The main drawbacks of the traditional seed production method are the seed tubers' high size, which drives up seed production costs, the ease with which viruses and other diseases can damage them, the slow rate of seed multiplication, and the lengthier time it takes to obtain breeder seed. There are various benefits to the invention and use of microtubers in potato farming. It is not necessary to acclimatize or move to a different medium when growing microtubers because they can be kept for up to a year and then transported straight to the market. By doing this, the field cycle needed to produce a sufficient amount of seed potatoes is greatly reduced, and a high quality of materials is guaranteed. Shoot tip and node or internodal explants, which have a survival probability of about 90%, are frequently used in the process of in vitro plant regeneration. Positive outcomes in stimulating shoot formation have been observed when Murashige and Skoog media enriched with benzyl amino purine (BAP) and kinetin is used. Furthermore, employing the MS medium has been reported to maximize the effectiveness of microtuber induction in potatoes. It's been found that exposing the plants to a 16-hour photoperiod and complete darkness can improve the effectiveness of microtuber induction even further. Ultimately, this review study offers an appropriate method for a successful pathway in the in vitro regeneration of potatoes by carefully examining prior scientific data.

First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.

The effectiveness of flow-through substrateless hydroponics in the adaptation of strawberry microplants

A new approach to the adaptation of test-tube strawberry microplants under flow-through hydroponics conditions was tested. The study was carried out on strawberry cultivars “Asia”, “Florence”, “Kimberly”. Murashige-Skoog medium was used for in vitro cultivation. During propagation, 1.0 mg•L-1 6-BAP was added to the medium, during elongation - 0.05 mg•L-1 6-BAP, and during rooting - 1.0 mg•L-1 IBA. Plants were grown in vitro in a light room at a temperature of 23±1°C, a 16-hour photoperiod and a light intensity of 5-6 klx. Flow hydroponics was used to adapt microplants. Plastic containers with dimensions of 0.6 x 0.4 x 0.04 m (volume 9 l), specially prepared for working in a hydroponic system, were taken. They were covered on top with non-woven polymer material, perforated with planting holes with a diameter of 3-4 mm according to a 4 x 4 cm pattern (150 pcs/box), into which plants were planted. This reduced labor costs for adaptation and ensured the efficiency of the operation to 86- 100% within 1 month.

Rhizoctonia solani AG1-IB, AG1-IC, and AG4-HGII cause bottom rot of field lettuce in Vermont.

Members of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) species complex cause bottom rot on lettuce (Latuca sativa) and yield losses up to 70% (Subbarao et al. 2017). Severe symptoms include necrosis, stem rot, and/or discoloration especially on the leaf midrib. In Vermont, vegetable farms are small (0.5-30 acres) and grow lettuce concurrently with other vegetable crops in the same field but the AG(s) that causes the disease in Vermont has not been determined. Isolates (n = 157) were collected from 31 fields with reported history of bottom rot between July 10 and October 8, 2019, across Addison, Caledonia, Chittenden, Franklin, Lamoille, and Orleans counties. Isolates were collected from lettuce tissue or potato (Solanum tuberosum), a common rotation crop, or uncropped soil baited using radish (Raphanus sativus). Pieces of tissue (5-10 mm) were cut from the leading margin of lesions, surface disinfested with 0.1% NaClO for 1 min followed by 2 rinses with sterile water, blotted dry, and plated onto acidified 2% water agar (0.085% lactic acid, pH 4.8). After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were examined for morphological characteristics including hyphal branching at ca. 90o angles, a septum near the branching point, multiple nuclei per cell, and lack of both clamp connections and conidia (Sneh et al. 1991). Colonies were white to dark brown, and some produced small sclerotia. Koch's postulates were performed by inoculating nine 8-week-old (9 leaf pairs) romaine lettuce plants (Johnny's Seeds, Winslow, ME, cv. Monte Carlo) per isolate. Isolates were grown on 2% potato dextrose agar for 1 week, from which a 5-mm agar plug was placed on the adaxial leaf surface at the base of a petiole. Plants were enclosed in a plastic bag to maintain high humidity and grown under a 16-hour photoperiod at 24 °C. Disease severity was rated 4 days after inoculation (0: healthy, 1: isolated lesions, 2: lesions across multiple petioles, and 3: systemic disease). Putative AG were determined by Sanger sequencing of the internal transcribed spacer (ITS) region using the ITS1F and ITS4B primer pair (758 bp) (Gardes and Bruns 1993). Contigs were assembled using CAP3 software (Huang and Madan 1999). Taxonomy was assigned to each OTU via the NCBI BLASTn database with criteria as 0.0 E and nucleotide match of at least 97%. Of the 10 isolates sequenced with sufficient coverage (735 to 784 bp alignment length) and definitive resolution (96.7 to 99.9% identity), 5 were putative AG 1-IB (Genbank Accession HG934430.1), 2 AG 1-IC (Genbank Accession AF354058.1), 2 AG 3 (Genbank Accession AF354064.1), and 1 AG 4-HGII (Genbank Accession AF354074.1). Fasta files and metadata are archived at 10.6084/m9.figshare.20301324, 10.6084/m9.figshare.20301375. Putative AG 1-IB was highly virulent on lettuce plants whether it originated from potato (mean 2.6) or lettuce (mean 1.3 to 3). AG 4-HGII and AG 1-IC isolated from lettuce and radish, respectively, were moderately severe (mean 1.4 to 2.2) on lettuce with identical symptoms. The two potato isolates (AG3) were not pathogenic on lettuce. Similarly, higher incidence of AG 1-IB is reported on lettuce in Quebec (Wallon et al. 2021), Ohio (Herr 1993), and Germany (Grosch et al. 2004). Because AG vary in their host range (Sneh et al. 1991), knowing the AG will inform management decisions such as crop rotation and weed control. This is the first report of the causal agent of bottom rot of lettuce or any AG of R. solani in Vermont.

Nutrient Solution Application of a Calcium-mobilizing Biostimulant Mitigates Tipburn without Decreasing Biomass of Greenhouse Hydroponic Lettuce

Lettuce tipburn is a physiological disorder characterized by marginal necrosis and curling of inner, younger leaves caused by localized calcium deficiency, especially in low evapotranspiration environments that restrict mass flow and thus calcium mobility. Severe tipburn negatively affects the marketability and quality of greenhouse-grown hydroponic lettuce. We aimed to assess the effectiveness of a chemical-based, calcium-mobilizing biostimulant for mitigating lettuce tipburn when applied in hydroponic nutrient solutions. Butterhead lettuce (Lactuca sativa ‘Rex’) was grown indoors under warm-white light-emitting diodes at a mean photosynthetic photon flux density of 300 μmol⋅m−2⋅s−1 for 11 days. Subsequently, we transplanted seedlings into deep-water-culture hydroponic trays in a greenhouse at an air temperature of 24.6 ± 1.2 °C, relative humidity of 76.2% ± 7.4%, and 20-hour photoperiod with supplemental lighting from high-pressure sodium lamps. The plants were grown in nutrient solutions with and without the biostimulant codenamed CC US-2105 at two concentrations (22 and 220 μL⋅L−1). Data were collected from plant samples at three harvests at 14, 21, and 28 days after transplant (DAT). At 14 DAT, there was no tipburn under any treatments. Compared with the control, the biostimulant at 22 μL⋅L−1 increased shoot dry mass by 31%. At 21 DAT, the biostimulant at 220 μL⋅L−1 eliminated tipburn, and the biostimulant increased shoot fresh weight by 28%, irrespective of the concentration. At 28 DAT, despite sufficient calcium in the whole plant and the remaining nutrient solution, severe tipburn still occurred in plants that did not receive the biostimulant (control). Compared with the control, the biostimulant at the higher concentration of 220 μL⋅L−1 decreased the tipburn rating by 88% and the number of leaves with tipburn by 85%, increased the plant diameter by 11%, increased the total leaf number by six, and accumulated higher levels of manganese and zinc. In contrast, these parameters remained unaffected at the lower biostimulant concentration of 22 μL⋅L−1. At 28 DAT, shoot biomass was unaffected by the biostimulant. In conclusion, the calcium-mobilizing biostimulant is an effective strategy to mitigate hydroponic lettuce tipburn without decreasing biomass accumulation in greenhouse conditions.

Embebição e germinação de sementes de chincuya (Annona purpurea Moc e Sessé Dunal) sob efeito de ácido giberélico e pH da água em imersão

Abstract: Germination is influenced by several factors, among them pH. Two experiments were conducted to determine the effect of imbibition-water pH on the water absorption curve of chincuya seeds and germination. The results were contrasted to the known effect of gibberellic acid imbibition. In the first year of fruit collection and evaluation, imbibition curves were constructed per treatment. The seeds of this evaluation were divided into two lots: the first at the initial moisture content (39.9 %) and the second at the moisture content of 34.1 % after three days of drying in the laboratory. The seeds were imbibed for 84 h in distilled water at three pH levels (5, 7, and 9) and kept in a water bath at 30 °C. In the second year of collection and evaluation, the pH treatments were repeated, and an imbibition treatment with gibberellic acid at 350 mg L-1 (pH 3.9) was added. A control treatment contained distilled water at pH 6.1. For both years, treated seeds were incubated on paper towels and placed under controlled conditions at a 12-hour photoperiod and alternating day and night temperatures (30 °C:25 °C). The three imbibition curves showed similar behavior and no statistical significance. Seeds with a higher moisture content did not gain weight after imbibition. The imbibition curves increased constantly in the seeds at 34.1 % moisture, and weight gain reached almost 46 % with the pH 9 treatment. However, there were no statistical differences among treatments. The highest rate of water absorption occurred in the first six hours in the open-air, dehydrated seeds, and after 72 hours, the weight of the seeds did not increase. Average daily germination kinetics indicated a slow and poor germination process. Gibberellic acid significantly promoted germination, causing 43 % germination. The absolute control reached 5.7 %, while the rest of the treatments did not promote germination.

Fungitoxic effect and phytochemical characteristics of Brazilian Cerrado weeds against Rhizoctonia solani and Macrophomina phaseolina fungi.

The use of natural products obtained from plants, for example, invasive plants, offers a variety of allelochemicals with fungicidal potential. With this in perspective, the objective was to evaluate the fungicidal potential of ethanolic extracts of Cerrado plants on Rhizoctonia solani and Macrophomina phaseolina. The ethanolic hydroalcoholic extract of the 12 plants identified as invaders in the Brazilian Cerrado was prepared (Anacardium humile Saint Hill; Baccharis dracunculifolia DC.; Cenchrus echinatus L; Commelina erecta L.; Erigeron bonariensis L.; Digitaria horizontalis Willd.; Digitaria insularis L.; Porophyllum ruderale Jacq. Cass; Richardia brasiliensis Gomes; Sida rhombifolia L.; Turnera ulmifolia L.; Smilax fluminensis Steud)) and phytochemical screening and determination of total phenols and flavonoids were performed. To evaluate the in vitro antifungal activity, the hydroalcoholic solutions at concentrations of 800, 1200, 1600, 2000, and 2400 µL 100 mL-1 were separately incorporated into BDA agar and poured into Petri dishes, followed by the mycelium disk of the fungus. As a control, two solutions were prepared, one ethanolic solution added to the BDA medium (2400 µg 100 mL-1) and the other with BDA medium only. They were poured into Petri dishes, followed by a 0.5 cm diameter disk of mycelium of the fungus, incubated (23±2 ºC), with a 24-hour photoperiod. Among the constituents found in the plants, 75% are phenolic compounds, 58.3% are cardiotonic heterosides, 50% are steroids, 33.3% are flavonoids, 16.7% are anthraquinones, and 8.3% are alkaloids, saponins, and reducing sugars. Out of the 12 species, only the extracts of C. erecta and R. brasiliensis were active for M. phaseolina and R. solani. Thus, it is concluded that the ethanolic extract of C. erecta has the fungicidal potential to control diseases caused by fungi that are soil inhabitants. Of the other species, A. humille, B. dracuncufolia, D. insulares, C. erecta, D. insulares, P. ruderale, and R. brasiliensis have natural fungitoxic potential because they stand out in the content of polyphenols efficient in reducing the mycelial growth of M. phaseolina and R. solani.

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 μm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 μl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

Potencial fitorremediador de mogno brasileiro (Swietenia macrophylla King) em exposição ao níquel: respostas anatômicas, bioquímicas e antioxidantes

Abstract The advancement and intensification of industrial and mining activities has generated a series of impacts on natural ecosystems, combined with the inappropriate use of agrochemicals and the erroneous disposal of electronic products, contributing to soil contamination with a diversity of chemical elements, including heavy metals. Due to this, this work aimed to evaluate the effect of increasing dosages of nickel on the anatomy, biochemistry and oxidative system of Brazilian mahogany (Swietenia macrophylla), a forest species from the Amazon, seeking to indicate the potential use of this species in phytoremediation programs. of soils contaminated with heavy metals. The seeds were grown under a constant temperature of 28°C, relative humidity (RH) of 90% with a 12-hour photoperiod for 43 days. The experimental design used was randomized blocks (DBC), with five treatments (0, 2, 4, 6 and 8 mg.L-1 of Nickel), with six replications. Data were subjected to analysis of variance (ANOVA) and means were tested for significant differences using the Tukey test at 5% significance. Changes in the anatomy of the different organs were observed, with differences in the cells in the central region of the leaf, the stem and the root. The concentration of total carbohydrates had no statistical differences with the application of nickel, however changes were observed in photosynthetic pigments, reducing sugars and sucrose as an adaptive form to nickel. The increase in nickel dosages was accompanied by the synthesis of ammonium, amino acids and proline in the root, while the synthesis of glycine was reduced. In the leaf, there was an increase in amino acids with an increase in metal, accompanied by a decrease in glycine. The plant antioxidant defense system was efficient in attenuating the toxic effects of ROS, with significant actions of CAT and SOD enzymes in the root, while the leaf had the main action of APX and CAT. The cultivation of mahogany plants can be advocated to mitigate Ni pollution in these areas, as this forest species has a particular characteristic of resistance to stressful conditions in contact with the heavy metal.

Characterization of intrapuparial development of Musca domestica (Diptera: Muscidae), under different temperatures in laboratory conditions.

Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), popularly known as "housefly", is a highly synanthropic species, with economic, medical-sanitary, veterinary, and forensic importance. It is able to cause damage to health, transmit pathogenic agents, cause infection in domestic animals, and, in its immature stage, cause secondary myiasis. The scavenging habit of its immature stages makes these flies pioneers in colonizing both human and animal carcasses, from the initial stages of corpse decomposition to the final stages. Intrapuparial development studies of all stages of the biological cycle of these insects help estimate pupal age, being useful to forensic entomology to aid in determining the minimum postmortem interval (minPMI). This study describes, morphologically, the external structures of the pupae, under temperatures of 23, 27, and 30 ± 1 °C aiming to identify the characteristics that define their developmental stages and estimation of the pupae age of M. domestica. The whole experiment was carried out under laboratory conditions, with relative humidity 60 ± 10% and 12 hours of photoperiod. The process of pupariation and pupation including pre-pupae phases were observed; larvae pupae apolysis; early cryptocephalic pupae; late cryptocephalic pupae; phanerocephalic pupae; pharate adult, discriminated by eye color (transparent eyes, pink eyes, and red eyes); and the emergency of adults, which occurred in the intervals of 162-180; 138-144, and 96-102 hr, respectively, being described throughout the metamorphosis of the external morphological characteristics of the pupal stage of M. domestica.

Establishment of Dendrobium wilsonii Rolfe in vitro regeneration system

Tissue culture processes have been established for Dendrobium wilsonii Rolfe, a valued ornamental and medicinal orchid. Pods aged 180 days served as seed sources. These seeds underwent surface sterilization and were then aseptically germinated on Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 Benzyl adenine (BA), 0.5 mg L−1 Naphthalene acetic acid (NAA), 20 g L−1 Sucrose (Su), 7 g L−1 Agar, 1 g L−1 Activated charcoal (AC), 1 g L−1 Peptone and 1 g L−1 Hyponex No.1 incubated in a growth chamber at 25.2°C with a 12-hour photoperiod and a light intensity of 2200 lx. Multiple shoot induction and proliferation was on MS medium in vitro produced shoots were rooted on MS medium with Hyponex No.1, 25 g L−1 Su, 1 g L−1 AC and 1.5 g L−1 Peptone. Peptone enhanced seedling growth and promoted clumped shoot development. The establishment of Dendrobium wilsonii Rolfe regeneration system provides a methodology for conserving germplasm resources and for commercial propagules production.