Sterilized Peat Moss Research Articles

Unraveling growth-promoting potential of plant beneficial actinobacteria on tropical bryophytes

Bryophytes are non-vascular plants with dominant gametophyte stage that play vital ecological roles in natural ecosystems. Unfortunately, their populations are currently in decline due to habitat destruction and various anthropogenic activities. The conservation efforts for bryophytes are hampered by their slow growth rates. This study aims to investigate the potential of actinobacteria to promote the growth of bryophytes. In this study, three plant growth-promoting actinobacteria, Dermacoccus abyssi MT1.1T, Micromonospora chalcea CMU55-4 and Streptomyces thermocarboxydus S3 were cultured in International Streptomyces Project medium 2 (ISP2) broth to obtain culture filtrates containing bioactive compounds for enhancing the growth of two bryophyte species, Physcomotrium sphaericum (C. Ludw.) Fürnr and Sphagnum cuspidatulum C. Müll. Interestingly, the incorporation of actinobacterial culture filtrates into 1/16 Murashige and Skoog (MS) medium yielded superior growth performance of P. sphaericum (C. Ludw.) Fürnr and S. cuspidatulum C. Müll, as observed from the thallus height, fresh weight, total chlorophyll contents, and total carotenoid contents compared to control groups. In addition, the inoculation of M. chalcea CMU55-4 on S. cuspidatulum C. Müll grown in sterile peat moss demonstrated the highest values for thallus height, fresh weight, dry weight, total chlorophyll content, and total carotenoid content. All actinobacteria successfully colonized the moss seedlings without any observable negative impacts, indicating beneficial interactions between actinobacteria and bryophytes. This research sheds light on the potential of harnessing plant beneficial actinobacteria to enhance the growth of bryophytes for conservation purposes.

First Report of a Root Lesion Nematode (Pratylenchus brachyurus) on Cassava in Taiwan.

The root lesion nematode, Pratylenchus spp., has a wide host range affecting many economically important crops (Castillo and Vovlas 2007). Cassava (Manihot esculenta Crantz) is an important food crop in several countries, commonly used for the material of bioethanol, animal feed, and starch extraction (Howeler 2014). Soil samples were collected from a mono-cropping cassava farm located in the Houbi district, Tainan city, in Southern Taiwan in October 2021 in a routine soil survey, and there is no obvious above ground symptoms. The cassava is a local cultivar, the sampled tuber did not have lesions, but small brown lesions were present on the roots. Nematodes were extracted using the modified Baermann funnel method (Tsay et al. 2004) for 24 h. Root lesion nematodes were the dominant genera in this sample, containing over 20 individuals per 100 cm3 of soil. Females of the root lesion nematodes were picked, and surface-sterilized with 2000 ppm malachite green for 30 sec and streptomycin for 30 mins. After sterilization, a single female was transferred onto the carrot discs to establish a pure line (Coyne et al. 2014). After 2 months, nematodes were extracted from that pure culture for morphometric, molecular identification, and pathogenicity tests. The female has a moderately slender body, a low and flat lip region, a sclerotized head frame, and a short ventral overlap of the esophagus, monovarial, prodelphic, and post-uterine sac. The tail is conical and the tip is rounded or flattened. The body measures of 20 females were: body length 564.43 μm (511 to 619 μm), stylet length 18.64 μm (18.1 to 19.5 μm), tail length 32.43 μm (27.1 to 38.5 μm), post uterine sac length 12.79 μm (9.41 to 16.9 μm), and V value 85.16% (84.1 to 86.6%). Values of a, b, c, and c' ratios were 22.32 (18.9 to 26.1), 6.11 (5.36 to 6.73), 17.55 (13.8 to 20.5), and 1.18 (0.92 to 1.5), respectively. All morphometric data were similar to the previous description of P. brachyurus (Castillo and Volvlas 2007). DNA was extracted from three nematodes of the pure cultures using Viagen DirectPCR lysis buffer, and used for PCR amplification of the 18S rRNA fragment and the D2-D3 expansion segment of 28S rRNA using primer sets D2A/D3B and 988F/2646R, respectively (Holterman et al. 2006; Subbotin et al. 2006). The sequence of 18S ribosomal RNA (OP020594) shared 99% similarity with the P. brachyurus sequence deposited in the GenBank database (KY424148), and the sequence of 28S rRNA (OP020593) also shared 99% similarity with several P. brachyurus sequences (e.g. KF712473, MG745329). Bayesian consensus trees, constructed from both 18S and 28S sequences revealed that the nematodes collected in this study are clustered together with P. brachyurus sequences from other countries(Subbotin, et al. 2008). Therefore, the nematodes collected from cassava were identified as P. brachyurus based on morphology, molecular data, and phylogenetic relationship. To determine the pathogenicity, three eight-week-old cassava plants (cv. TMS 60444) were planted in 12-cm-diameter pots filled with 600 cm3 of sterile peat moss: sand (1:1, W: W) and inoculated with 50 nematodes containing different stages. Two plants treated with water were used as the mock control. Seventy-five days after inoculation, nematodes in the soil were recovered using the modified Baermann funnel method for 24 h, and the nematodes inside the root were stained with acid fuchsin. The average reproduction factor (final population/initial population) was 3.93, thus confirming cassava as a host of P. brachyurus. P. brachyurus was previously reported on peanuts and bananas in Taiwan, and wasn't a dominant species in the field. Finding this nematode on this cassava farm suggests this nematode might have a wider distribution than expected.

Using marsh organs to test seed recruitment in tidal freshwater marshes.

PremiseSeed recruitment niches along estuarine elevation gradients are seldom experimentally field‐tested under tidal regimes of the Pacific Northwest of North America. Addressing this knowledge gap is important to better understand estuary restoration and plant community response to sea level rise.MethodsGermination was tested in marsh organ mesocosms across an elevation gradient (0.5–1.7 m above mean sea level). Seeds were sown on sterile peat moss, and the tops of pipes were secured with horticultural “frost cloth” to ensure no experimental seeds were washed out and no new seeds were introduced. The trials tested artificial and overwinter chilling regimes, as well as the presence and/or absence of a near‐neighbor transplant.Results Carex lyngbyei had significant elevation‐driven germination after overwinter and artificial chilling. Schoenoplectus tabernaemontani had near‐significant germination across elevation after overwinter chilling, and germination in the absence of competition was significantly greater than with a near‐neighbor transplant.Discussion Carex lyngbyei had the highest germination rate at higher elevations, which suggests restricted seed recruitment potential and required clonal expansion to extend into lower marsh elevations. Identifying species‐specific recruitment niches provides insight for restoration opportunities or invasive species monitoring, as well as for estuary migration under sea level rise.

First Report of Root-Knot Nematode Meloidogyne enterolobii onCockscomb (Celosia argentea var. cristata) in Taiwan.

Cockscomb (Celosia argentea) is commonly found in subtropical and temperate zones of Africa, South America and South East Asia, and is a popular ornamental plant in the family Amaranthaceae. Cockscomb has been known to contain antiviral proteins, betalains, and anthocyanin, which can be applied in beneficial ways (2). In September 2020, a cockscomb plant (Celosia argentea var. cristata) showing typical galling root symptoms likely infected by root-knot nematodes (Meloidogyne sp.) was collected from a garden in Taichung, Taiwan, and a quick exam of several individuals using MK7F/R primers (7) indicating they were M. enterolobii. Nematode population was established from a single egg mass and was later used for species identification and pathogenicity tests. Five perineal patterns of mature females from the single female population show round to oval shapes with weak lateral lines. Dorsal arches are moderate to high, almost squared, with the smooth ventral striae. Second-stage juveniles are vermiform and have a slender tail, tapering to rounded tip with distinct hyaline region at the tail terminus. Morphological measurements of 28 J2s revealed body length = 457.2 ± 20.6 (416.1-506.9) μm, body width = 16.0 ± 2.0 (13.4-20.3) μm, stylet length = 14.7 ± 0.5 (13.9-15.9) μm, dorsal gland orifice to the stylet base = 4.0 ± 0.5 (2.0-4.8) μm, and tail length = 56.0 ± 3.8 (47.4-60.3) μm. Female perineal patterns and morphometric data are similar to the original description of Meloidogyne enterolobii (9). DNA purified from approximately 1500 juveniles using GeneMark Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan) was used to amplify 18S rDNA fragment, D2-D3 expansion segments of 28S rDNA, and a COII region on mtDNA with primer sets 1A/MelR, D2A/D3B, and C2F3/1108, respectively (4,5,6). The 18S rDNA sequence (OK076893) of this study shares 99.94% nucleotide identity with those of M. enterolobii isolated from the United States (KP901058) and China (MN832688). D2D3 sequence of haplotype 1 (OK076898) shows 100% identity to those of M. enterolobii from China (MT193450) and Taiwan (KP411230). Sequence of haplotype 2 (OK076899) shows 99.86% identity to those of M. enterolobii from the United States (MN809527) and China (MN269945). Sequence of the COII region (OK086042) show 99.86% identity to that of M. enterolobii from China (MN269945). Phylogenetic trees of the three gene sequences were plotted following Ye et al.(10), revealing that the newly described root-knot nematode on Cockscomb is grouped with other M. enterolobii isolates. DNA fragment amplified by primer sets Me-F/R(3) and MK7F/R specifically targeting of M. enterolobii yielded 236 bp and 520 bp, respectively. Pathogenicity tests were assayed, from July to September 2021, on three-week-old nematode-free cockscomb plant directly germinated from seeds of SkyStar® (ASUSA SPIKE SEEDS, Taipei, Taiwan) planted in a 10.5 cm diameter pot filled with 600 ml sterilized peat moss: sand (1:1, v/v) soil in a 28℃walk-in chamber. Nematode eggs were extracted using 0.05% NaoCl as described by Vrain(8), and cockscomb plants (n=3) were inoculated by adding 6000 eggs (10 eggs/ cm3). Cockscomb plants treated with water were used as mock controls. Rf value of the inoculated plants were determined by the method of Belair and Benoit (1) 45 days after inoculation, and the average was 4.13. No galls were observed on the roots of control plants. The results confirmed that cockscomb is the new host of M. enterolobii. To the best of our knowledge, this is the first report of M. enterolobii on Celosia argentea var. cristata in Taiwan.

First Report of Fusarium incarnatum-equiseti Species Complex Causing Leaf Spot on Rockmelon (Cucumis melo L.) in Malaysia.

Rockmelon, (Cucumis melo L.) is an economically important crop cultivated in Malaysia. In October 2019, severe leaf spot symptoms with a disease incidence of 40% were observed on the leaves of rockmelon cv. Golden Champion at Faculty of Agriculture, Universiti Putra Malaysia (UPM). Symptoms appeared as brown necrotic spots, 10 to 30 mm in diameter, with spots surrounded by chlorotic halos. Pieces (5 x 5 mm) of diseased tissue were sterilized with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) and incubated at 25°C for 7 days with a 12-h photoperiod. Nine morphologically similar isolates were obtained by using single spore isolation technique and a representative isolate B was characterized further. Colonies were abundant, whitish aerial mycelium with orange pigmentation. The isolates produced macroconidia with 5 to 6 septa, a tapered with pronounced dorsiventral curvature and measured 25 to 30 μm long x 3 to 5 μm wide. Microconidia produced after 12 days of incubation were single-celled, hyaline, ovoid, nonseptate, and 1.0 to 3.0 × 4.0 to 10.0 µm. Morphological characteristics of the isolates were similar to the taxonomic description of Fusarium equiseti (Leslie and Summerell 2006). Genomic DNA was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA). To confirm the identity of the fungus, two sets of primers, ITS4/ITS5 (White et al. 1990) and TEF1-α, EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify complete internal transcribed spacer (ITS) and partial translation elongation factor 1-alpha (TEF1-α) genes, respectively. BLASTn search in the NCBI database using ITS and TEF-1α sequences revealed 99 to 100% similarities with species of both F. incarnatum and F. equiseti. BLAST analysis of these in FUSARIUM-ID database showed 100% and 99% similarity with Fusarium incarnatum-F. equiseti species complex (FIESC) (NRRL34059 [EF-1α] and NRRL43619 [ITS]) respectively (Geiser et al. 2004). The ITS and TEF1-α sequences were deposited in GenBank (MT515832 and MT550682). The isolate was identified as F. equiseti, which belongs to the FIESC based on morphological and molecular characteristics. Pathogenicity was conducted on five healthy leaves of 1-month-old rockmelon cv. Golden Champion grown in 5 plastic pots filled with sterile peat moss. The leaves were surface-sterilized with 70% ethanol and rinsed twice with sterile-distilled water. Then, the leaves were wounded using 34-mm-diameter florist pin frog and inoculated by pipetting 20-μl conidial suspension (1 × 106 conidia/ml) of 7-day-old culture of isolate B onto the wound sites. Control leaves were inoculated with sterile-distilled water only. The inoculated plants were covered with plastic bags for 5 days and maintained in a greenhouse at 25 °C, 90% relative humidity with a photoperiod of 12-h. After 7 days, inoculated leaves developed necrotic lesions similar to the symptoms observed in the field while the control treatment remained asymptomatic. The fungus was reisolated from the infected leaves and was morphologically identical to the original isolate. F. equiseti was previously reported causing fruit rot of watermelon in Georgia (Li and Ji 2015) and China (Li et al. 2018). This pathogen could cause serious damage to established rockmelon as it can spread rapidly in the field. To our knowledge, this is the first report of a member of the Fusarium incarnatum-F.equiseti species complex causing leaf spot on Cucumis melo in Malaysia.

In vitro Multiple Shoot Regeneration from Petunia hybrida

An efficient plant regeneration protocol was developed from in vitro germinated seeds of Petunia hybrida an ornamentally important plant in the family Solanaceae. Shoot tip and node explants of Petunia hybrida were cultured on MS basal medium supplemented with different concentrations and combinations of Benzyl amino purine (BAP), 1-Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Gibberellic acid (GA3). The highest shoot length was obtained from MS medium supplemented with 1 mg/l BAP + 1 mg/l NAA. The highest shoot number (3 shoots/explant) were obtained from MS medium supplemented with 0.6 mg/l BAP + 0.5 mg/l IBA. The isolated shoots were transferred to MS basal medium supplemented with different concentrations of GA3 ranging from 0.05, 0.2, 0.5 and 1 mg/l for shoot elongation. The highest shoot length (5.75 cm) was recorded from the MS medium supplemented with 0.2 mg/l GA3 +0.2 mg/l BAP. Rooting of regenerated shoots were achieved on MS medium supplemented with 0.1-1 mg/1 IBA and NAA. The regenerated shoots with well developed roots were successfully acclimatized and established in pots containing sterilized peat moss and grown under laboratory conditions with 70% survival rates.

Intergeneric Hybrid from Jatropha curcas L. and Ricinus communis L.: Characterization and Polyploid Induction.

Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry for its oil. However, the cultivation of J. curcas L. is faced with numerous challenges unlike the cultivation of Ricinus communis L. (2n = 2× = 20), a closely related species. The generation of an intergeneric hybrid between J. curcas L. and R. communis L. was investigated. Intergeneric hybrids were produced by hand crossing. Immature embryos were rescued, in vitro, from the hybrid seeds and cultured on an enriched Murashige and Skoog (MS) medium for a month. The plantlets produced were grown in sterile peat moss in plastic pots and covered with polyethylene for 30 days, after which they were transferred into cement potted soil. The hybridity and the genuineness of the hybrids were successfully confirmed using randomly amplified polymorphic DNA (RAPD) markers. The number of branches, stem diameter, and leaf size of the F1 hybrids were similar to those of J. curcas L. while the plant height was similar to that of R. communis L. Young hybrids were treated with various concentrations (0%, 0.3%, 0.4%, and 0.5%) of colchicine to induce polyploids. The calli (JR6) treated with 0.3% colchicine recorded the highest tetraploid cell percentage (38.89%). A high tetraploid cell percentage (>50%) is significant in overcoming the problem of sterility after hybridization.

Prioritization ofMalusaccessions for collection cryopreservation at the USDA-ARS National Center for Genetic Resources Preservation

The USDA-ARS National Plant Germplasm System maintains a grafted collection of apple accessions representing 48 taxa in Geneva, NY. Dormant buds of many of these accessions have been routinely cryopreserved at the USDA-ARS National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, CO. In the standard procedure, dormant buds are sent to NCGRP in mid-winter. Scions are cut into 35 mm bud sections and desiccated at -5°C to a moisture content of 25 to 30% (fresh weight basis). Desiccated single-bud sections are then sealed into polyolefin tubes containing 10 to 12 sections each, slow cooled at -1°C h-1 to -30°C, held at -30°C for 24 h, and then placed into the vapor phase of liquid nitrogen (LNV) for long term storage. For viability testing, the buds from one polyolefin tube are rehydrated at 2°C in moist, sterile peat moss and grafted onto rootstocks. For most accessions, this left 3 polyolefin tubes of Malus buds in long-term storage for each accession. For this analysis, successfully cryopreserved accessions were defined as those that have a total of 19 or more predicted viable buds remaining in LNV at NCGRP. Of the 2291 accessions currently cryopreserved at NCGRP, 2052 accessions meet this requirement. Criteria were established to prioritize the apple accessions that are either inadequately backed up at NCGRP or have not yet been processed. These criteria include the likelihood of success in cryopreserving the Malus taxon, the vulnerability of the field trees, the number of viable buds currently stored in LNV, and previous failures in response to the standard procedure.

FIRST REPORT OF FUSARIUM WILT OF ALBIZIA JULIBRISSIN CAUSED BY FUSARIUM OXYSPORUM f. sp. PERNICIOSUM IN SPAIN

In 2014, Albizia julibrissin trees located in gardens at Logrono municipality (La Rioja, Northern Spain) showed wilt symptoms, including defoliation, internal black streaks and, finally, tree death. Necrotic wood samples were surface disinfected for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g l–1), and incubated at 25°C in the dark. Fusarium colonies were consistently isolated and transferred to Spezieller Nahrstoffarmer agar (SNA). Ten days after incubation at 25oC, all isolates were identified as F. oxysporum, based on the presence of short monophialides, abundant microconidia produced in false heads (length 7.5 to 12.5 μm, average 9.20 μm) and chlamydospores, and sparse, usually three-septate, macroconidia (length 21.25 to 40 μm, average 29.70 μm) (Leslie and Summerell, 2006). The translation elongation factor 1-alpha gene (TEF) region of the isolates was amplified with primers EF1 and EF2 (O’Donnell et al., 1998). Sequences showed 99% identity with a sequence of F. oxysporum f. sp. perniciosum (FJ985413). One sequence was deposited into GenBank (accession No. KU050689). Pathogenicity of isolates GIHF-019 and GIHF-021, was determined on one-year-old A. julibrissin plants grown in sterile peat moss, which were inoculated by watering the roots with 100 ml of a conidial suspension (106 conidia ml–1). Plants were maintained at 25oC in a growth chamber under a photoperiod of 12 h. Two months after inoculation all inoculated plants wilted and the fungus was reisolated from them. To our knowledge, this is the first report of F. oxysporum f. sp. perniciosum in Spain.

Evaluation of grapevine rootstocks against soilborne pathogens associated with trunk diseases

The susceptibility of the grapevine rootstocks most commonly used in Spain to black-foot (Ilyonectria liriodendri and Dactylonectria macrodidyma-complex) and petri disease (Cadophora luteo-olivacea, Phaeomoniella chlamydospora and five species of Phaeoacremonium) pathogens was evaluated. Rooted cuttings of rootstocks 110R, 1103P, 140Ru, 161-49C, 196-17C, Fercal and SO4 were inoculated with black-foot pathogens by dipping their roots in conidial suspensions (5×105 conidia mL-1), planted in pots containing sterilized peat moss and placed in a greenhouse. After four months of incubation, root disease severity index and dry weights of shoots and roots were recorded for each plant. Regarding petri disease pathogens, one-year-old grapevine cuttings of five rootstocks (41B, 140Ru, 161-49C, 1103P and 110R) were vacuum-inoculated with spore suspensions (106 conidia mL-1) of the fungal species and planted in two fields. After four months, the proportion of vines that sprouted in spring was visually determined. At the end of the growing season, dormant plants were uprooted, washed, and assessed for undried shoot weight. Then, the stem of each grapevine cutting was transversally split at 10 cm from the base of the plant to estimate the percentage of vascular tissue discoloured on a scale of 0 to 4. All rootstocks inoculated with Ilyonectria and Dactylonectria species were affected by the disease in some degree, the rootstock 110R being the most susceptible to black-foot. Petri disease pathogens caused a significant reduction of sprouting and shoot weight, as well as a significant increase of disease severity percentage in all grapevine rootstocks with the exception of 161-49C. Both 110R and 140Ru were the most susceptible. Grapevine rootstocks showed different levels of disease resistance, being the rootstock 110R the most susceptible to both diseases and the 161-49C rootstock the most tolerant to petri disease infection.

Biocontrol of Pythium aphanidermatum by the cellulolytic actinomycetes Streptomyces rubrolavendulae S4

Streptomyces rubrolavendulae (Yen) S4, isolated from a termite mound in Kanchanaburi, Thailand, was used to control the seedling damping-off disease of the horticultural plant Joseph's coat caused by Pythium aphanidermatum (Edson) Fitz. In a previous study, S. rubrolavendulae S4 was shown to strongly inhibit the growth of P. aphanidermatum on potato dextrose agar. This study investigated the modes of the antagonistic action of S. rubrolavendulae S4 on P. aphanidermatum. On carboxymethyl cellulose medium, S. rubrolavendulae S4 produced the highest cellulase activity of 65 U/ml in 5 days. The S. rubrolavendulae culture supernatant, with and without heat treatment, was tested for fungal growth inhibition using an agar well-diffusion method. The inhibitory effect was retained with the heat inactivated culture supernatant, indicating that the inhibition was derived from an antifungal compound rather than an enzyme produced by S. rubrolavendulae S4. The parasitic activity of S. rubrolavendulae S4 upon P. aphanidermatum was demonstrated by light and scanning electron micrographs. Joseph's coat (Amaranthus tricolor) seedlings were grown in peat mosses inoculated with S. rubrolavendulae S4 and P. aphanidermatum. In pots containing S. rubrolavendulae S4, the proportion of healthy seedlings was significantly increased. This was equivalent to the effect of the fungicide metalaxyl, and similar to the results from sterilized peat moss. Hence S. rubrolavendulae S4 can be used as an effective biological control agent in Joseph's coat cultivation.

First Report of Cylindrocladiella parva and C. peruviana Associated with Black-foot Disease of Grapevine in Spain

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] μm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] μm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Koch's postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe.

In vitro propagation of Rosa hybrida L. cv. Al-Taif Rose plant

In this study, a protocol for in vitro propagation of Rosa hybrida L. cv. Al-Taif Rose was established using nodal segments harboring axillary buds as explants. In vitro stages of shoot initiation, multiplication and elongation were performed. Explants were cultured on solid Murashige and Skoog medium (MS) supplemented with different concentrations of benzyl aminopurine (BAP, 1, 2 and 3 mg/l) in combination with 1 mg/l kinetin (Kn). Effect of different concentrations and combinations of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on root formation of shoots were studied. The highest percentage of shoot initiation (85%) was observed on MS medium containing 2 mg/l BAP + 1 mg/l Kn, whereas maximum average number of multiplied shoots (2.7) was produced on MS medium with 3 mg/l BAP + 1 mg/l Kn. Highest average number of elongated shoots (26.7) was noticed on MS medium containing 1 mg/l BAP and 1 mg/l Kn. For rooting, highest percentage (66.7%) of rooted shoots was obtained on MS medium supplemented with 2 mg/l IBA. Plantlets with 4 to 5 roots of 3 to 5 cm length were successfully transferred to pots containing sterile peat moss for acclimatization. Keywords: Rosa hybrid L. cv. Al-Taif Rose, axillary bud explants, in vitro propagation, multiplication, acclimatization

Antifungal activity of some actinomycetes isolated from Riyadh soil, Saudi Arabia: An evaluation for their ability to control Alternaria caused tomato blight in green house pot trial)

A collection of 105 strains of actinomycetes isolated from the soils of Riyadh, Saudi Arabia were evaluated for their ability to inhibit plant pathogenic Fusarium oxysporum, Alternaria alternata, Aspergillus niger, and Aspergillus flavus in vitro. About 61.90% isolates were able to inhibit the growth of A. alternata, followed by 61.8, 52.24 and 50.9% inhibiting A. flavus, F. oxysporum and A. niger respectively. Subsequently, these isolates were tested for their ability to control Alternaria blight on tomato plant (Lycopersicon lycopersicum) grown in sterilized peat moss soil. Broth and crude extract of all except, DS-8(32), DS-6(48) and DS-6(33) isolates of actinomycetes have shown effect on plant disease severity. Over all analysis of data showed that strain AS-2(29) was the most effective isolate.Broth and crude extract of this strain had shown maximum zone of inhibition against F. oxysporum (11 and 18 mm respectively) and A. alternata (15 and 17 mm respectively). Plants treated with this strain were healthy and green and only a few older leaves showed the blight symptom and growth was found at par with control plants. However, strain DS-6(33) has not shown any antifungal activity and plants succumbed to infection of A. alternata, whereas, broth of DS-8(24) and DS-1(23) showed strong antifungal activity against A. alternata (20 and 19 mm zone of inhibition). However, crude extract of these strains has not shown any antifungal activity and plants treated with these strains were healthy and only older leaves were blighted. Extract but not broth of DS-1(20) showed strong antifungal activity against F. oxysporum and A. alternata (15 and 16 mm respectively) and the growth of the plant was similar to that of the control plants in pot trial strains varied in their antifungal activity against one or both plant pathogenic fungi. Based on the cultural, morphological and physiological characteristics, most of the strains were identified as the different isolates of the genus Streptomyces. In the further study, Streptomyces species as biological control agents would be offered a much needed alternative to the use of synthetic agrochemicals. Key words: Actinomycetes, screening antifungal activity, A. alternata, green house pot trial, scanning electron microscope, tomato blight disease.

First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Spain.

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) μm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) μm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) μm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) μm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) μm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain.

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) μm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) μm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain.

First Report of Fusarium Wilt Caused by Fusarium oxysporum f. sp. basilici on Ocimum minimum in Portugal.

Ocimum minimum L. (fine-leaved basil) is cultivated along the Douro Litoral Region in Portugal. In June 2009, a disease was observed in fine-leaved basil fields in three geographically separated locations: Maia, Rio Tinto, and São Mamede Infesta. Affected plants showed wilt symptoms, chlorotic leaves, and stem necrosis. Discolored vascular tissue was observed when the stems were cut longitudinally. For isolation, crown and stem sections (10 cm long) were surface disinfected for 1 min in 1.5% NaOCl and washed twice with sterile distilled water. The sections were cut longitudinally and small pieces of discolored vascular tissue were plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g liter-1). Plates were incubated at 25°C in the dark. Fusarium colonies were consistently isolated from symptomatic plants sampled from the three different locations and transferred to PDA and Spezieller Nährstoffarmer agar (SNA) culture media for morphological species identification (2). After 10 days of incubation at 25°C, all isolates were identified as F. oxysporum. A PCR-based assay was conducted with nine single-spored isolates (F2, F3, F4, F7, F8, F9, F10, F11, and F13) using the F. oxysporum f. sp. basilici specific primer pair, Bik 1 and Bik 2 (1). A single DNA fragment of 382 bp was amplified in all isolates, which confirmed the identification of F. oxysporum f. sp. basilici. Pathogenicity of all nine isolates was determined on 2-month-old fine-leaved basil seedlings growing in sterile peat moss. Plants were inoculated by watering the roots with 20 ml of a conidial suspension (106 conidia ml-1) harvested from 3-week-old cultures grown on PDA. Thirty-six replicates (each one in individual pots) for each isolate were used, with an equal number of control plants. Plants were maintained in a greenhouse at 15 to 20°C. Within 2 weeks of inoculation, all inoculated plants wilted and exhibited severe leaf and stem necrosis. The fungus was reisolated from vascular tissues of the crown area and the stems of symptomatic seedlings, fulfilling Koch's postulates. Control plants remained healthy. To our knowledge, this is the first report of F. oxysporum f. sp. basilici infecting fine-leaved basil in Portugal.

IN VITRO PROPAGATION OF POTENTILLA FULGENS HOOK (BAJRADANTI) – A HIGH VALUE MEDICINAL HERB FOR COMMERCIAL CULTIVATION

A rapid in vitro clonal propagation protocol is standardized for Potentilla fulgens, a high value medicinal herb of Indian Himalayas. The best promising results were obtained by culturing the tender petiole segments for adventitious shoot proliferation in Murashige and Skoog basal medium supplemented with a combination of benzyl adenine (BA, 1.5 mg/l) and indole acetic acid (IAA, 0.5 mg/l). The best rooting (92.3%) was obtained while culturing the in vitro raised shoots in MS medium modified with IAA (1.5 mg/l). Rooted plantlets were hardened in sterilized peat moss in plastic pots. Spray of quarter strength MS salts solution on alternate days is found reducing the mortality of plants considerably during the hardening process. As a result it is possible to obtain 30-35 rooted plant from a single explant within three months, and hence is useful for commercial cultivation.

Requirement for particular seed‐borne fungi for seed germination and seedling growth of Xyris complanata, a pioneer monocot in topsoil‐lost tropical peatland in Central Kalimantan, Indonesia

AbstractHawaii yellow‐eyed grass (Xyris complanata: Xyridaceae) inhabits infertile, acidic peat soil in the rainy tropical zone in Southeast Asia. This monocot plant produces a large number of dormant seeds in order to make a large deposit to seed bank in the soil. Under laboratory conditions, surface‐sterilized X. complanata seeds are rarely able to germinate on sterilized peat moss bed; they require inoculation with either seed epiphytic or soil fungi to facilitate active seed germination. In the present study, three different genera of seed epiphytic fungi were isolated, and two common fungal genera, Fusarium sp. (strain R‐1) and Penicillium sp. (strain Y‐1), were found to promote seed germination of X. complanata. In sterile peat moss beds, the germination‐stimulating fungi also showed growth‐promoting effects on X. complanata seedlings. These results suggest that the seed germination‐promoting fungi likely function as genuine partners for X. complanata in tropical open peat lands.

Effect of Soilless Potting Media and Water Management on Development of Fungus Gnats (Diptera: Sciaridae) and Plant Growth

Coconut coir dust is being marketed as a soilless medium substitute for sphagnum peat moss that inhibits fungus gnat (Bradysia sp.) development. However, little information is available on the effects of coconut coir dust on Bradysia sp. In a laboratory study we examined the effect of substituting coconut coir dust for peat moss, with or without a food source, on the development of fungus gnats. An average of less than one adult emerged when 20 fungus gnat eggs were provided with pure or sterilized peat moss or coconut coir. A significantly higher number of adults (11.5-13) emerged when a food source of 1 g of yeast was added to either soilless potting medium type. The adults required up to 10 fewer days to emerge when food was provided, compared to sterilized and pure media, except for the pure peat moss. In a greenhouse study examining the effects of coir and peat at different textures and different moisture levels on fungus gnat survival, there were significant differences at the different levels of moisture. There was a higher population of larvae in the coarse medium containing peat. In the coir-based media, the fine-textured medium had the highest population level of fungus gnats. There were no significant effects on fungus gnat populations among the different levels of moisture within a medium type. However, there was a tendency for lower populations in the most moist and the driest media and the highest survival in the media that were maintained at 52.5% moisture. Plant growth was best in the media with the lowest number of fungus gnats (coarse coconut coir dust-based and fine and medium peat-based media). These results suggest that it is possible to select growing media that minimize fungus gnat populations, while optimizing plant growth. However, contrary to claims made by growing media producers, coconut coir dust does not necessarily inhibit fungus gnat development.