EFFECT OF DIFFERENT NATURAL MIXED MEDIA ON THE BIOLOGICAL CHARACTERISTICS OF THE GREEN LACEWING Chrysoperla carnea (STEPHENS)

Peanut (Arachis hypogaea L.) is one of the most profitable upland crops, yielding 10,711 tonnes in an area of 4,062 ha in the Republic of Korea (Ministry of Agriculture, Food and Rural Affairs 2023). In September 2023, dark gray spots surrounded by yellow halos were observed on the peanut leaves over an area of 880 m2 at the National Institute of Crop Science (35°50'31.4"N 127°02'41.0"E), with a disease incidence up to 80%. Early symptoms appeared as small, brown, circular or irregular spots that enlarged and were surrounded by chlorotic halos. Leaf cuttings (5 mm x 5 mm) from five symptomatic plants were surface-sterilized with 70% EtOH for 1 min, followed by 1% NaClO for 1 min, and rinsed 3 times with sterile water. The pieces were placed on Potato Dextrose Agar (PDA) and incubated at 25 °C in the dark for 3 days. Three isolates obtained by single-spore isolation were designated as F23025, F23026, and F23027. Two isolates, F23025 and F23026 were deposited in the Korean Agricultural Culture Collection (https://genebank.rda.go.kr) under the accession numbers 410722 and 410723. Fungal colonies were initially white and turned sooty gray after 5 days. Conidia were unicellular, brown to black, and spherical or sub-spherical with 6.8 μm to 14.3 μm (mean = 11.1 μm ± 1.8, n = 50). The morphology of the three isolates was identical and showed the same characteristics as Nigrospora oryzae (Ellis 1971; Hudson 1963). For molecular identification, the Internal Transcribed Spacer (ITS) region (GenBank accession PP388306 and PP574448), beta tubulin (PP397027 and PP580108), and translation elongation factor 1- ɑ (PP397028 and PP580109) of isolates F23025 and F23026 were amplified and sequenced with primers of ITS5/ITS4, Bt2a/Bt2b, EF1-727F/EF2, respectively and showed high identity of 99.62% (530 bp/532 bp), 100% (384/384), and 99.79% (475/476) with N. oryzae strain LC2693 (GenBank accessions KX985994, KY019471, and KY019299, respectively). Multilocus sequence analysis showed isolates F23025 and F23026 were on the same clade with N. oryzae strain LC2693. To determine the pathogenicity to peanut, a conidial suspension (1 x 106 conidia/mL) was sprayed onto leaves of five 3-week-old plants 'Sewon' grown in pots, while sterile distilled water was sprayed onto two plants used as negative control. Sprayed plants were placed in a dew chamber at 25℃ for two days and grown in a growth chamber at 25℃ and 80% of relative humidity with a 16L:8D cycle. Two weeks later, dark spots with chlorotic halos appeared only on leaves sprayed with conidia, and no symptoms on leaves sprayed with sterile distilled water. The pathogenicity test was repeated three times, and each time the pathogen was re-isolated and identified by ITS sequence, thus fulfilling Koch's postulates. Nigrospora species are cosmopolitan, and some species have a wide host range as plant pathogens. Recently, two species of the genus Nigrospora, N. sphaerica and N. aurantiaca, were reported to cause peanut leaf blight in China (Liu et al. 2020; He et al. 2023). To the best of our knowledge, this is the first report of N. oryzae causing leaf spot to A. hypogaea L. in the Republic of Korea. As identifying new pathogens and registering fungicides to control them are important for the continued cultivation of peanut, this report will help in that endeavor.

A field experiment during the spring season of the 2023 was Conducted in the agricultural fields of the Agricultural Research Department – Ministry of Agriculture – Abu Ghraib. The aim of this research was to investigate the effect of seed stimulation treatments with hydrogen peroxide and sowing dates on yield traits of Sorghum cultivars . The experiment was Arranged with the split split plots by using a Randomized Complete Block Design (RCBD) with three replicates and three factors. The main plots were allocated for three planting dates (11/3/2023 and 25/3/2023 ). The sub plots included three varieties of white corn (Al-Khair, Buhooth - 70, and Rabih). The sub sub plots also incorporated a seed stimulation treatment with hydrogen peroxide at a concentration of 9 ml l-1 , additionally, two control treatments were included: seed priming with distilled water only and dry seeds. The results showed that sorghum seeds stimulated with hydrogen peroxide gave highest averages of weight of 500 grain and grain yield . The second planting date 25/3/2023 gave highest averages of number of grains in the head (head - 1) and grain yield . Buhooth-70 cultivar gave highest averages of weight of 500 grain and grain yield. The interaction of seeds stimulated with hydrogen peroxide and planted on first planting 11/3/2023 showed significant superiority in traits weight of 500 grain and grain yield. Buhooth-70 cultivar seeds stimulated with hydrogen peroxide give the highest averages for traits weight of 500 grain and grain yield .

First Report of Bacterial Dry Rot of Mango Caused by <i>Sphingomonas sanguinis</i> in China

During 2013, mango (Mangifera indica) producers of the most important growing area of Iran (Hormozgan province), reported extensive damages to panicles of mango trees of cv. Lengra, consisting of necrosis of flowers and pedicels. A fungus was isolated from affected panicles and its morphological traits were determined on colonies grown on Synthetic Nutrient Agar (SNA) medium as described by Bensch et al. (2012). Conidia 3-6×5.5-20 μm in size were fusiform with visible scars extensions. A portion of the translation elongation factor 1-α gene was amplified (Bensch et al., 2012) and sequenced (GenBank accession No. KT247610). A BLAST search showed 99% similarity with GenBank sequences belonging to Cladopsorium tenuissimum. Four healthy inflorescences were selected on a mango tree and, after disinfection by spraying 1% sodium hypochlorite and thoroughly washing with sterile distilled water, were sprayed with a spore suspension. Inoculum was prepared by rinsing a 7-day-old culture on PDA with sterile water and adjusted to 106 spores/ml. The inoculated inflorescences were kept covered with thin polythene bags for two days. Four control panicles on the same experimental tree were similarly disinfected and kept covered with thin polythene bags, after spraying with sterile distilled water. Symptoms resembling those observed in the field appeared on inoculated panicles from which the pathogen was re-isolated. Control treatments developed no symptoms. To our knowledge, this is the first report of C. tenuissimum causing inflorescence infection on magno in Iran.

First Report of Cape Gooseberry Scab Caused by <i>Cladosporium exasperatum</i> in Thailand

Muskmelon (Cucumis melo L.) is an economically important fruit crop in China. In September 2018, fruit rot was observed on approximately 20% of muskmelon fruits in Harbin, Heilongjiang Province, China. Brown water-soaked lesions were observed on the fruit side in contact with soil initially, which gradually extended to most of or the entire fruit. Internal decay was observed with white to dark brown mycelium on the fruit surface. Diseased muskmelon tissues were surface disinfested with 1% NaOCl for 3 min, 70% ethanol for 10 s, and then washed three times with sterile water. The disinfested tissues were cut into 1-cm pieces and placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 1 week. Isolations were done on 10 fruit fragments, and the same fungus was obtained. The cultures were purified using the hyphal-tip technique and used for morphological and molecular analyses. Morphological characteristics were observed on 1-week-old PDA cultures grown at 28°C. The aerial mycelium changed from white to light yellow, and the back of the plate turned pale brown with time. Microconidia were single celled, hyaline, nonseptate, ovoid, and 8.5 to 10.6 × 3.2 to 4.4 μm. Hyaline macroconidia (mostly three-septate) were slightly curved at the apex and ranged from 19.4 to 35.2 × 3.8 to 7.7 μm. Chlamydospores with thick, roughened walls were abundant in clumps or chains, ellipsoidal or subglobose. Primers ITS1/ITS4 (White et al. 1990) and EF-1/EF-2 (O’Donnell et al. 2000) were used to amplify and sequence the internal transcribed spacer (ITS1-5.8S-ITS2) and translation elongation factor 1-α (TEF1) region. ITS and TEF1 gene sequences were deposited in NCBI GenBank nucleotide database with accession numbers MH910492 and MH920853, respectively. BLASTn analysis showed 99% nucleotide sequence identity with Fusarium incarnatum-equiseti species complex (FIESC). The TEF1 gene sequence of isolate NEAU-TG1 was compared with sequences in the FUSARIUM-ID database (Geiser et al. 2004), which indicated that the pathogen was most closely (99% identity) related to phylogenetic species within the FIESC. Pathogenicity of these isolates was confirmed by following Koch’s postulates. Ten muskmelon fruits of cultivar Xiangfei were surface disinfested with 2% NaOCl for 2 min and rinsed with sterile distilled water three times. Spores were produced on PDA for 7 days at 28°C and washed with sterile distilled water; the concentrations were adjusted to 1 × 10⁶ spores/ml using a hemocytometer. Ten muskmelon fruits were inoculated by injecting the suspension (2 ml, 1 × 10⁶ spores/ml) and covered with plastic bags for 24 h, and another 10 surface-disinfested fruits treated with sterile distilled water were used as a control. The fruits were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber at 25°C with 12-h day/night cycle for 8 days. All inoculated fruits showed symptoms identical to those observed in the field. No disease occurred on the controls. The pathogen was reisolated from diseased fruits, and species identification was confirmed by the morphological and molecular method described above. Members of the FIESC can cause disease on plants, such as Allium cepa, Festuca arundinacea, Morchella importuna, and Cucumis trigonus. Furthermore, the members of the FIESC have been reported to produce type A and B trichothecene mycotoxins that cause toxicosis in humans and animals (Leslie and Summerell 2006; O’Donnell et al. 2009). To our knowledge, this is the first report of fruit rot caused by a member of the FIESC on muskmelon in China. This pathogen presents a threat to muskmelon production, and its accurate identification is necessary to develop effective management strategies.

Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable.

Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable.

To ascertain the efficacy of different fungicides, the experiments were conducted in a laboratory. Slide germination technique (SGT) was employed to test the fungicides for inhibition of spore germination. Spore suspension of fungus was prepared by adding 10 ml of sterile distilled water to 10 days old well grown fungal slants. The concentration was adjusted to 50 spores per microscopic field at 10 x power of magnification by further dilutions with sterile distilled water. Equal volumes of the given fungicide respectively for each concentration and the spore suspension were thoroughly mixed. One drop of this was placed in sterilized cavity slide. These slides were placed in moist chambers from a pair of petriplates carrying wet blotting paper to maintain adequate humidity and incubated at 25 +_ 10 c for 24 hours. Each treatment was replicated three times for each concentration. In case of control only sterile water was added instead of fungicide. Observations were recorded after 24 hours Germinated and ungerminated spores were counted separately and the per cent spore germination was calculated.

This study evaluated the efficacy of chlorinated water, sodium hypochlorite solution, sodium chloride solution and sterile distilled water in eliminating pathogenic bacteria on the surfaces of raw vegetables. Lettuce vegetables were dipped in different concentrations of chlorinated water, sodium hypochlorite solution, sodium chloride solution and sterile distilled water for 1,2,3,4 and 5 minutes and analyzed microbiologically. Treatments of the vegetables with chlorinated water, sodium hypochlorite solution, salt water and distilled water did not eliminate the pathogens, but reduced their populations. The bacterial loads were reduced by 38.18 – 69.83% (chlorinated water), 23.74 – 69.13% (sodium hypochlorite solution), 31.62 – 79.0% (salt water) and 14.95 – 42.59% (sterile distilled water). The coliform loads were reduced by 41.2 – 68.9% (chlorinated water), 22.2 – 55.6% (sodium hypochlorite solution), 36.0 – 90.0% (salt water) and 22.2% - 40.0% (sterile distilled water). This suggests that very high concentrations of salt water could be useful in reducing pathogenic microorganisms in fresh produce.

First Report of <i>Fusarium culmorum</i> Causing Crown Rot on Wheat in Jordan

This research aimed to findout efficient sterilization method and MS media to germinate dragonfruit seeds in vitro , conducting at Biotechnology Laboratory, Agriculture Faculty, Tadulako University, using factorial completely randomized design. The first factor was sterilization technique (S), i.e : S1 (The sliced fruit was washed with sterile distilled water three times, then rinsed in Bayclin (household bleach containing 5.25% NaOCl) 5% for 15 minutes, followed by washing in sterile distilled water three times. S2 (The seeds were taken from the fruit meat, then rinsed in 5% Bayclin for 15 minutes, then washed with sterile distilled water three times. S3 (The seeds were taken from the fruit meat, then rinsed in 15% Bayclin for 15 minutes, then washed with sterile distilled water three times. The second factor was the strength of MS media (M), i.e full strength MS (M1) and half strength MS for macro and micro nutrients (M2). Each treatment combination consisted of 35 seeds, and was replicated three times. Germination responses were observed as time to germinate, germination percentage and percentage of opened cotyledone seedlings. All datas were subjected to Analysis of Variance and the mean differences among the treatments were analyzed using Honest Significant Difference (HSD) at the level of 1%. The results showed that removing seed pulp, prior to rinsing the seeds in 15% Bayclin for 15 minutes followed by washing in sterile aquadest three times, and cultured in half MS produce the fastest and highest seed germination of 99.05% with 92.38% opened cotyledon seedlings after 2 weeks in culture. Key Words: Dragonfruit, germination, in vitro, MS strength, sterilization.

Radish (Raphanus sativus L.), which belongs to Brassicaceae, is widely cultivated throughout China. The 'Shawo' radish is renowned for its crispness, sweetness, and juiciness, and predominantly cultivated as a fruit in Tianjin. In November 2023, black rot on radish tubers was observed in several commercial fields on different farms (approximately 10 ha total, with distance of 2-3 km), with disease incidence of 40%-50% in Xiqing District (116.97°E, 39.09°N), Tianjin city, China. Infected plants were stunted and grew yellow leaves, the radish tubers turned blackish brown and emitted a foul odor, eventually rotting completely inside. No fungal mycelium or spores were found on infected tissues by microscopic examination. To identify the causal agent, four radish tubers with rot symptoms were collected from four distinct affected fields belonging to four different farms. Small pieces of tissue (5 × 5 mm) were excised from the margin of diseased and healthy tuber with sterile scalpel blades, soaked in 75% alcohol for 30 s, rinsed three times in sterile distilled water, then ground, suspended in sterile distilled water, spread onto nutrient agar (NA) plates and incubated at 28°C for 24 h. Individual colonies on NA plates appeared oval, slightly convex, translucent with a shiny surface and regular edges. Three independent isolates (LB2, LB3 and LB4), each derived from a tuber in three different fields, were identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991). Sequences (accession nos. PQ066487, PQ066488 and PQ066489) had 99.29% homology to that of Pectobacterium parvum (CP087392.1). Housekeeping genes icdA, mdh, pgi, and proA were amplified from all three strains with primer pairs of icdA400F/icdA977R, mdh86F/mdh628R, pgi815F/pgi1396R and proAF1/proAR1 (Ma et al. 2007), resulting in 100% sequence identity across all the strains. The sequences of representative strain LB4 were uploaded to GenBank with accession numbers PP066861, PP066862, PP066863, and PP066865, respectively. Phylogenetic analysis showed that LB2, LB3 and LB4 clustered with P. parvum type strain s0421T (Pasanen et al. 2020). All the three isolates were negative for utilization of citrate, and no color change on Simmons' citrate agar within 72 h inoculation. The pathogenicity test of the three strains was carried out by inoculating 20 µL of each bacterial suspension (108 CFU/mL), which was prepared in sterile distilled water, onto conical wounds. The conical wounds, with a depth of 3 cm and a top diameter of 4 mm, were created by using a sterile 200 μL pipette tip pricked on the surface of five healthy 'shawo' radish tubers. Sterile distilled water was used as a control. Tested radish tubers were incubated in a glass cabinet maintained at 28°C with 90% relative humidity with the inoculated area left open. The pathogenicity test was repeated three times per strain. All the suspension inoculated 'shawo' radish tubers exhibited softened epidermis, blackish brown and severe internal decay three days after inoculation, similar to the symptoms observed in the field, while control radish tubers without symptoms. P. parvum was reisolated from the infected 'shawo' radish tubers and confirmed by sequencing comparition described above, thus fulfilling Koch's postulates. Pectobacterium parvum was first isolated and described as a causal agent of Brassica rapa subsp. chinensis in China by Pasanen et al. 2020. Then, P. parvum was reported causing potato stem rot in Hebei and Inner Mongolia Autonomous Region, China (Wang et al. 2022; Wu et al. 2023). To our knowledge, this was the first report of P. parvum causing radish (shawo cv.) tuber rot in China as well as in the world. This pathogen of P.parvum requires further research on epidemiology and disease management options against its potential impact on local plantation of shawo radish.

This research aimed to findout efficient sterilization method and MS media to germinate dragonfruit seeds in vitro , conducting at Biotechnology Laboratory, Agriculture Faculty, Tadulako University, using factorial completely randomized design. The first factor was sterilization technique (S), i.e : S1 (The sliced fruit was washed with sterile distilled water three times, then rinsed in Bayclin (household bleach containing 5.25% NaOCl) 5% for 15 minutes, followed by washing in sterile distilled water three times. S2 (The seeds were taken from the fruit meat, then rinsed in 5% Bayclin for 15 minutes, then washed with sterile distilled water three times. S3 (The seeds were taken from the fruit meat, then rinsed in 15% Bayclin for 15 minutes, then washed with sterile distilled water three times. The second factor was the strength of MS media (M), i.e full strength MS (M1) and half strength MS for macro and micro nutrients (M2). Each treatment combination consisted of 35 seeds, and was replicated three times. Germination responses were observed as time to germinate, germination percentage and percentage of opened cotyledone seedlings. All datas were subjected to Analysis of Variance and the mean differences among the treatments were analyzed using Honest Significant Difference (HSD) at the level of 1%. The results showed that removing seed pulp, prior to rinsing the seeds in 15% Bayclin for 15 minutes followed by washing in sterile aquadest three times, and cultured in half MS produce the fastest and highest seed germination of 99.05% with 92.38% opened cotyledon seedlings after 2 weeks in culture. Key Words: Dragonfruit, germination, in vitro, MS strength, sterilization.

Giant philodendron (Philodendron giganteum Schott) is cultivated in Thailand and has become an important ornamental houseplant with great economic value. During the rainy season in July 2022, anthracnose disease on this plant was observed at a nursery in Saraphi District, Chiang Mai Province (18°40'18" N, 99°03'17" E), Thailand. The area investigated was approximately 800 m². The disease incidence was estimated at above 15% according to the total number of plants (220 plants). The disease severity of each plant was between 25 and 50% of the necrotic lesion on the leaf. Initially, symptoms with brown spots, appeared on leaves, gradually becoming enlarged, elongate, 1 to 11 cm long by 0.3 to 3.5 cm wide, irregular, sunken, dark brown, with a yellow halo surrounding each lesion. Then, the diseased leaves eventually withered and died. Leaf pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface sterilized in 1% NaClO for 1 min, 70% ethanol for 30 s, and rinsed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. After three days of incubation, pure fungal colonies were purified by a single hyphal tip method on PDA (Korhonen and Hintikka 1980). Two fungal isolates (SDBR-CMU471 and SDBR-CMU472) with similar morphology were obtained. Fungal colonies on PDA were white and 38 to 40 mm in diameter after 3 days of incubation at 25 °C, then grayish white with cottony mycelia, the reverse side pale yellow after one week of incubation. Both isolates produced asexual structures on PDA. Setae were brown with 1 to 3 septa, 50 to 110 × 2.4 to 4.0 µm, with a cylindrical base, and acuminate tip. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 9.5 to 35 µm long (n = 50). Conidia were single-celled, straight, hyaline, smooth-walled, cylindrical, ends rounded, guttulate, 9.1 to 19.6 × 3.5 to 5.6 µm (n = 50). Appressoria were brown to dark brown, oval to irregular, smooth-walled, 5 to 10 × 5 to 7.5 µm (n = 50). Morphologically, both fungal isolates resembled members of the Colletotrichum gloeosporioides species complex (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), β-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OQ699280, OQ699281; act: OQ727122, OQ727123; tub2: OQ727124, OQ727125; CAL: OQ727126, OQ727127; GAPDH: OQ727128, OQ727129). Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum likelihood phylogenetic analyses demonstrated that both isolates were identified as C. siamense with 100% support. In a pathogenicity test, leaves of healthy plants were surface sterilized with a 0.1% NaClO solution for 3 min, rinsed three times with sterile distilled water. After being air-dried, a uniform wound (5 pores, 3 mm in width) was made at the equator of each leaf using aseptic needles. Conidial suspensions were collected from two-week-old cultures and suspended in sterile distilled water with 0.05% Tween-20. Fifteen microliters of the conidial suspension (1 × 106 conidia/ml) were placed on wounded attached leaves. As well, wounded control leaves were mock inoculated with sterile distilled water. Ten replications were conducted for each treatment and experiments were repeated twice. The inoculated plants were stored in a greenhouse at conditions of 25 to 30°C and 75 to 85% relative humidity. After 14 days, all the inoculated leaves showed disease symptoms, brown lesions with yellow halos, whereas control leaves remained asymptomatic. The pathogen C. siamense was consistently re-isolated on PDA from the inoculated tissues to complete Koch's postulates. Colletotrichum siamense has been reported as a causal agent on a wide range of host plants in Thailand and throughout the world (Farr and Rossman 2021; Jayawardena et al. 2021). Prior to this study, C. endophytica, C. karsti, C. orchidearum, C. philodendricola, and C. pseudoboninense were identified as causal agents of anthracnose on philodendrons (Xue et al. 2020; Zhang et al. 2023). However, anthracnose caused by Colletotrichum species on giant philodendron (P. giganteum) has not been previously reported. Thus, we propose C. siamense as a new causal agent of anthracnose disease on giant philodendron. This study provides information for further investigation into the epidemiology and management of this disease. Moreover, further investigations should be carried out in other philodendron growing areas of Thailand in order to specifically search for this pathogen.