Identification and Characterization of a Streptomyces albus Strain and Its Secondary Metabolite Organophosphate against Charcoal Rot of Sorghum

Streptomycesalbus strain CAI-21 has been previously reported to have plant growth-promotion abilities in chickpea, pigeonpea, rice, and sorghum. The strain CAI-21 and its secondary metabolite were evaluated for their biocontrol potential against charcoal rot disease in sorghum caused by Macrophomina phaseolina. Results exhibited that CAI-21 significantly inhibited the growth of the pathogen, M. phaseolina, in dual-culture (15 mm; zone of inhibition), metabolite production (74% inhibition), and blotter paper (90% inhibition) assays. When CAI-21 was tested for its biocontrol potential under greenhouse and field conditions following inoculation of M. phaseolina by toothpick method, it significantly reduced the number of internodes infected (75% and 45% less, respectively) and length of infection (75% and 51% less, respectively) over the positive control (only M. phaseolina inoculated) plants. Under greenhouse conditions, scanning electron microscopic analysis showed that the phloem and xylem tissues of the CAI-21-treated shoot samples were intact compared to those of the diseased stem samples. The culture filtrate of the CAI-21 was purified by various chromatographic techniques, and the active compound was identified as “organophosphate” by NMR and MS. The efficacy of organophosphate was found to inhibit the growth of M. phaseolina in the poisoned food technique. This study indicates that S.albus CAI-21 and its active metabolite organophosphate have the potential to control charcoal rot in sorghum.

First report of quinoa anthracnose caused by Colletotrichum nigrum and C. truncatum in the United States

Quinoa (Chenopodium quinoa Willd.) is increasingly produced outside its native Andean range. In September 2019, stem lesions were observed on all six plants of quinoa accessions PI 510547 (25% severity) and PI 596293 (75% severity) in a demonstration plot in Ames, IA. Lesions were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips and contained setose acervuli. Fungi were isolated from both accessions following disinfestation with 70% ethanol and plating onto ½ acidified potato dextrose agar (APDA) and V8 medium. Isolates were examined morphologically. On V8 medium, isolate CQ1 produced sparse, flat, gray mycelia with profuse sclerotia and hyaline, aseptate, cylindrical conidia (n= 50, mean: 21.0 (range: 19.2-24) by 4.3 (2.4-4.8) µm); isolate CQ2 produced fluffy, gray to dark gray mycelia with profuse sclerotia and acervuli and hyaline, aseptate, falcate conidia (n =50, 26.8 (24-31.2) by 2.4 µm). Direct hyphal PCR was used to amplify ITS (ITS1/ITS4), ACT (ACT-512F/ACT-783R), GAPDH (GDF1/GDR1), CHS-1 (CHS-79F/CHS-234R), and TUB2 (T1/Bt-2b, CQ1 only) (Fu et al. 2019, Liu et al. 2013), and products were sequenced bidirectionally (MT772082-3, MT786524-30). A maximum likelihood tree was generated in MEGA X (Kumar et al. 2018) from a multiple sequence alignment of vouchered CBS isolates (Liu et al. 2013) and CQ1 was identified as Colletotrichum nigrum. CQ2 sequences showed 99-100% similarity to Colletotrichum truncatum sequences in Genbank (MN581860, MK675238, MF682518, MK118057). Koch’s postulates were completed once with two isolates of each species grown on V8 medium under 12 hours of near UV light for two weeks. Greenhouse conditions were a 12 hr day/night cycle and temperature range of 26-30° C. Approximately 5 mL of mycelium on agar medium was sterilely removed and macerated in 6 mL of sterile distilled water. Non-inoculated medium was macerated in sterile water as a control. Forty-day old quinoa PI 634920 were inoculated by making three, 2-3 mm incisions between the cotyledons and first true leaves with a sterile razor blade. Next, 500 µL of slurry was placed on 2.54 cm2 of sterile cheesecloth and placed against the wound and wrapped with Parafilm. Six plants were inoculated per isolate and control. After two weeks, sunken, bleached to tan areas extended past wound sites of inoculated plants. No discoloration or sunken tissue was observed on control plants. Plants were tented with plastic film for one week. Acervuli were observed on C. truncatum- and C. nigrum-inoculated stems, and sclerotia were observed on C. nigrum-inoculated stems. Stems were surface disinfested with 10% bleach and plated onto ½ APDA. Colony morphologies of isolated fungi matched those of original inoculum for inoculated plants. Colletotrichum spp. were never isolated from control plants. When stems were inoculated, approximately 100 µL of slurry was also placed on 3-5 detached quinoa leaves in Petri dishes with moistened blotter paper and incubated for 48 hours at 25° C. Brownish, circular lesions developed on leaves inoculated with either species, but no lesions developed on control-slurry leaves. Colletotrichum spp. cause disease in quinoa relatives including spinach (Kurt 2015), beets (Gourley 1966) and amaranth (Wu 2001). This is the first description of Colletotrichum spp. causing stem lesions on quinoa in the United States. This disease may emerge in new quinoa production regions and may cause yield losses due to lodging.

Management of Dry Root Rot in Chickpea (Cicer arietinum L.) Caused By Macrophomina phaseolina by Utilizing Host Plant Resistance, Fungicides and Bioagents

Background: For the management of soil borne disease like dry root rot of chickpea caused by Macrophomina phaseolina, by using fungicides alone is not feasible due to environmental and health hazards. Hence integrated management of the disease by using resistant varieties, fungicides and bio-control agents is the best alternative. So the present study was aimed to identify resistant varieties, best fungicide and bioagent for management of dry root rot in chickpea. Methods: Two hundred and twelve genotypes were screened using blotter paper technique for identifying resistant genotypes for dry root rot. The experiment on management of dry root rot was conducted during Kharif and Rabi of 2013-14 using a susceptible chickpea variety JG-11 with 14 treatments including control with 3 replications. Result: Of two hundred and twelve chickpea genotypes screened for host plant resistance against Macrophomina phaseolina by blotter paper technique only one genotype ie. PBG-5 showed moderately resistant reaction. Among fourteen treatments including fungicides and bioagents imposed for the management of dry root rot, seed treatment with tebuconazole @ 2 g/kg recorded lowest per cent disease incidence of 9.43, with a highest yield of 722.81 kg/ha compared to untreated control which recorded the highest per cent disease incidence (40.10) with a lowest mean yield of 362.02 kg/ha.

Mycofloral study of cultivated maize seed in district Poonch Azad Jammu and Kashmir

The current study was carried out on the prevalence of mycoflora associated with maize seeds using blotter paper method. Maize seed samples were collected from six different locations of district Poonch, Azad Jammu and Kashmir. Objective of this study was to determine the fungi associated with maize seeds. A total of seven species of fungi were isolated. Penicillium spp., Fusarium spp., Aspergillus niger, A. flavus, Fusarium spp., Fusarium oxysporum and Pythium spp., were identified. Davigali had the infection percentage of 72 % in all locations. Occurrence frequency and type of fungi isolated varied with location. Prevalence of pathogenic fungi with maize seeds of district Poonch was found variable. Resistant varieties of maize, maintaining temperature, relative humidity and their treatment is suggested to reduce disease and increase yield.

Bionomics of the African Apefly (Spalgis lemolea) as A Potential Natural Enemy of the Papaya Mealybug (Paracoccus marginatus) in Tanzania

The African apefly (Spalgis lemolea Druce) is a potential natural enemy of the papaya mealybug (Paracoccus marginatus Williams and Granara de Willink). We studied the life history of apeflies in the laboratory at a temperature of 25–27 °C and a relative humidity of 55%–65% under a 12 h photoperiod condition. The papaya mealybugs and apefly larvae were collected from papaya plants in Tengeru, Arusha, Tanzania. The papaya mealybugs were introduced and allowed to multiply on potted sprouting potato plants in screened cages. In order to study the life cycle and predation of apeflies, an apefly egg was placed on an open screen-covered petri dish containing a moist blotter paper and observed for larva emergence. After the apefly larva emergence, a mixture of mealybug eggs (up to 1500), nymphs (200–250) and adults (100–150) was introduced in the petri dish each day and the consumption rate by the apefly larvae was quantified until the larvae reached pupal stage. Then, the apefly adults were collected and put into cages 30 cm × 30 cm × 30 cm containing cotton wool soaked in water, for observation of pre-mating, mating, egg-laying and life span. Results indicate that the apefly completed its life cycle in 23 days. The mean development period of the eggs was four days and the development period for the larva, pre-pupa, and pupa was nine, one and ten days respectively. The adult apefly emerged after 9 days of the pupa stage with mean body lengths of 10.1 ± 0.4 mm and 11.0 ± 0.8 mm for the males and females, respectively. The female laid an average of 68 eggs in groups of two to seven at different sites after 4–5 days of emergence. In this study one apefly larva was capable of consuming about 1983 ± 117, 123 ± 6 and 80 ± 9 papaya mealybug eggs, nymphs and adults respectively during larval stage. These results reveal the predatory potential of the apefly in suppressing the population of papaya mealybugs under field conditions.

Identification of 1-Butyl-Lysergic Acid Diethylamide (1B-LSD) in Seized Blotter Paper Using an Integrated Workflow of Analytical Techniques and Chemo-Informatics

The rapid dispersion of new psychoactive substances (NPS) presents challenges to customs services and analytical laboratories, which are involved in their detection and characterization. When the seized material is limited in quantity or of a complex nature, or when the target substance is present in very small amounts, the need to use advanced analytical techniques, efficient workflows and chemo-informatics tools is essential for the complete identification and elucidation of these substances. The current work describes the application of such a workflow in the analysis of a single blotter paper, seized by Swedish customs, that led to the identification of a lysergic acid diethylamide (LSD) derivative, 1-butyl-lysergic acid diethylamide (1B-LSD). Such blotter paper generally contains an amount in the range of 30–100 ug. This substance, which is closely related to 1-propionyl-lysergic acid diethylamide (1P-LSD), seems to have only recently reached the drug street market. Its identification was made possible by comprehensively combining gas chromatography with mass spectrometry detection (GC–MS), liquid chromatography coupled with high-resolution tandem MS (LC–HR-MS/MS), Orbitrap-MS and both 1D and 2D nuclear-magnetic-resonance (NMR) spectroscopy. All the obtained data have been managed, assessed, processed and evaluated using a chemo-informatics platform to produce the effective chemical and structural identification of 1B-LSD in the seized material.

Seed-borne infestation and management of Alternaria Species in mustard seed at Chitwan district, Nepal

Alternaria blight in mustard cause heavy damage to the foliar parts resulting into poor growth and development of plants and thereby reduces seed yield. Inorder to manage Alternaria infestation in mustard seed for yield maximization, an experiment was conducted in the plant protection laboratory in Nepal Polytechnic Institute, Bharatpur, Chitwan in 2018, using Completely Randomized Design (CRD) with four replicates. Five treatments i.e.Uthane M-45 (2g/kg), Neem powder (3 g/kg), Bojho powder (4g/kg), Trichoderma harzianum, (107 Conidia/mL) and control (without treatment) were used for seed treatment of local variety of Mustard. Four hundred seeds for each treatment (25 seeds in each petriplate) were plated using triple layers of moistened blotter paper in petriplate and incubated at (27±20C) for 2 days and followed deep freezing for 24 hrs. Data of disease incidence and seed germination were recorded in 3, 7 and 10 days after incubation (DAI). Seedling vigor and seedling weight were also recorded at 5 DAI. The percentage of Alternaria spp incidence on seeds at different DAI showed highly significant with respect to different treatments. Application of Uthane M-45 and T. harzianum significantly reduced the seed-borne infection of Alternaria spp. as compared to control. At 3 DAI Uthane M-45 completely checked the pathogen however, only 4% and 5% disease incidence was observed in 7 DAI and 10 DAI respectively. Bio control agent Trichoderma harzianum was found next best alternative after Uthane M-45 to control disease. Plant extract Bojho powder and Neem powder were found better than control to check the disease. Furthermore, highest germination (76%) was observed in Trichoderma treatment whereas, lowest germination was found in control.

Screening Chickpea Genotypes for Resistance to Rhizoctonia bataticola in Controlled Conditions

Among the various biotic stresses, dry root rot caused by Rhizoctonia bataticola is becoming severe in most chickpea growing regions of India where the crop is grown under rain fed conditions causing 30-40 per cent yield loss. In this context, 520 chickpea genotypes were screened in the laboratory condition using Blotter Paper Technique to study the reaction of the genotypes to the Rhizoctonia bataticola and to identify resistance source for the disease. Among 520 genotypes, three were resistant viz., PG 06102, BG 2094 and IC 552137; 21 were moderate resistant viz., IC 15167, IC 2867, JAKI 9218, ICC 9023, ICC-14346, IC-269768, PG 01103, Pule 9801, KGD 120, NbeG 28, WR 315, IC-269488, IC 552198, IC 552178, IC 552132, IC 552320, IC 552214, IC 552232, CLH 29, IC 552102, IC 552224; 76 were moderate susceptible, 337 were susceptible and the rest eighty three were highly susceptible for dry root rot. The identified resistant genotypes may serve as potential donors in chickpea resistance breeding programme for dry root rot.

Germination and osmotic adjustment in Salvia hispanica L. (Lamiaceae) seedlings under water and thermal stress

Salvia hispanica seedlings have stood out for their potentialities in nutrients for agri-food industries. Nevertheless, studies related to this species are still scarce, especially with respect to agronomic factors that enable its propagation and development. Hence, this study aimed to evaluate germination, vigor and osmotic adjustment of S. hispanica seeds and seedlings subjected to water and thermal stress. The experimental design was completely randomized, with treatments arranged in factorial scheme, corresponding to five levels of polyethylene glycol (PEG 6000) (0.0; -0.1; -0.2; -0.3 and -0.4 MPa) and five temperatures (20, 25, 30 and 20-30 °C), with four replicates of 50 seeds in each treatment. Germination test was set on blotter paper, moistened with PEG 6000 solutions at the previously mentioned potentials, under 8-h photoperiod. The following variables were analyzed: germination, germination speed index, seedling shoot length, seedling root length, seedling dry matter and contents of chlorophyll, carotenoids, amino acids, proline and sugars. Polyethylene glycol levels of -0.4 MPa at all temperatures and -0.3 MPa at 30 °C and 20-30 °C enable germination and vigor of S. hispanica seedlings. S. hispanica seedlings are able to perform osmotic adjustment under water stress conditions up to the level of -0.3 MPa, when grown from seeds germinated at temperatures of up to 25 ºC.