Moist Chamber Research Articles

An annotated checklist slime molds (Myxomycetes = Myxogastrea) of western Kazakhstan

Winter-cold arid regions of western Kazakhstan were surveyed for myxomycetes for a period of 20 years. A total of 3228 records belonging to 111 species from 31 genera and 10 families are provided in an annotated checklist. The checklist contains data on the localities, habitats, substrates, methods of collection and voucher numbers of specimens deposited in the mycological herbarium (LE) of the V.L. Komarov Botanical Institute of the Russian Academy of Sciences. Additionally the bibliographic references of the myxomycete species findings in the study area are given. Due to the very arid climate of the region, 2911 specimens (ca. 90%) were obtained from 1653 moist chamber cultures prepared with samples taken from bark of living plants, litter and the weathered dung of herbivorous animals. Only 317 specimens of myxomycetes were collected directly in the field, mostly in woody artificial plantations. The lowest species diversity was observed in habitats with halophytic vegetation, where on average only 1–2 species were recorded per moist chamber culture. Only <em>Perichaena depressa</em> and <em>P. liceoides</em> were common under such conditions. The highest diversity of myxomycetes was observed in the intrazonal woody communities of the steppe zone, which are usually associated with river valleys and artificial woody plantations. In these habitats lignicolous species occurred: <em>Amaurochaete atra</em>, <em>Arcyria obvelata</em>, <em>Cribraria cancellata</em>, <em>Lamproderma scintillans</em>, <em>Lycogala epidendrum</em>, <em>Metatrichia vesparia</em>, <em>Oligonema flavidum</em>, <em>Stemonitis axifera</em>, <em>S. fusca</em>, <em>S. herbatica</em>, <em>S.pallida</em>, <em>Symphytocarpus confluens</em>, and <em>Trichia contorta</em>. However, the apparently most common species of myxomycetes in the studied area are associated with litter or bark: <em>Badhamia foliicola</em>, <em>B. spinispora</em>, <em>Didymium anellus</em>, <em>D. difforme</em>, <em>D. trachysporum</em>, <em>Echinostelium colliculosum</em>, <em>Fuligo cinerea</em>, <em>Licea denudescens</em>, <em>L. nannengae</em>, <em>L. parasitica</em>, <em>Macbrideola oblonga</em>, <em>Pericaena depressa</em>, <em>P. corticalis</em>, <em>P. liceoides</em>, <em>P. vermicularis</em>, <em>Physarum cinereum</em>, and <em>Ph. pseudonotabile</em>. Among substrate types, species diversity and richness decrease from wood over ground litter to bark, and dung of herbivorous animals. Shannon diversity and species richness reached maximum values in the intrazonal and artificial woody communities, whereas treeless sagebrush desert and dry steppe communities and, halophytic vegetation had the most depauperate yet most specific myxomycete assemblages. Assemblages associated with these vegetation types displayed a high level of similarity to those of myxomycete assemblages from other arid regions of Kazakhstan and Central Asia. In contrast, assemblages of the artificial woody plantations in the study region displayed a high level of similarity to those of boreal forest regions of Siberia for which data exist, but differed from the assemblages documented from treeless desert and steppe regions of Eurasia.

Myxomycetes of boreal forests of the Laplandskiy State Nature Biosphere Reserve (Kola Peninsula, Russia) Yuri

Northern taiga forests and subalpine plant communities of the Laplandskiy State Nature Biosphere Reserve (Kola Peninsula, Russia) were surveyed for myxomycetes. A total of 1675 specimens of myxomycete fruit bodies (sporocarps) were registered, among them 1584 records from field collections and 92 obtained from 210 moist chamber cultures of ground litter, bark of living trees, wood, and weathered dung of moose and willow ptarmigan. Most of 125 taxa (124 morphospecies and one variety) representing 34 genera were recorded only in the field (104 taxa from 32 genera), but some were exclusively obtained from moist chamber cultures (8 taxa from 5 genera). All of the recorded species are new for the Laplandskiy Reserve. Species numbers decreased among the four studied forest associations along the elevation and mositure gradient, and the Shannon index showed a similar trend: spruce forest (PICa; 84 taxa, H’=3.8), spruce-peat moss forest (PICb; 70, 3.5), dry spruce-pine forest (PIN; 62, 3.7), subalpine birch forest (SB; 30, 2.7). The estimated completeness of the survey according to the Chao1 estimator was 66%, indicating that most of the more common species should have been recovered. The trend among forest associations runs mainly parallel to diversity: PICa and PICb 83%, PIN 47%, SB 57%. The myxomycete assemblage of dry coniferous forests is the most distinctive among the three forest types and shows the highest number of indicator species. The overall degree of specialization of myxomycetes is higher for substrate type than for forest associations. Among substrate types, species diversity and richness increase from litter over bark to wood.

Metodologia para avaliação da resistência à rizoctoniose em germoplasma de meloeiro

The intensive cultivation of melon, mainly in the Northeast of Brazil, has favored the occurrence of root diseases such as rhizoctonia, caused by the fungus Rhizoctonia solani Kuhn. The use of resistant cultivars is one of the most efficient strategic measures for integrated management of diseases in this crop. It is necessary to use an efficient inoculation method to evaluate rhizoctonia in melon germplasm with the aim of identifying sources of resistance. The objective of this study was to develop an efficient method to inoculate the pathogen to proceed in selection of R. solani resistant melon germplasm. Five experiments were conducted under greenhouse conditions to define the adequate container, growth environment, substrate for inoculum production, aggressiveness of the isolates, inoculum density, manner of inoculation, phenological stage of the plant, and time for evaluation after inoculation. The results obtained showed that for selection of R. solani resistant melon germplasm, the use of seedlings with roots cut during transplanting to pots is recommended. The pots should contain sand with organic inoculum obtained from rice grains at the concentration of 150 mg.kg-1 of soil, associated with inoculation through a puncture in the plant stem by an infested toothpick, without the need for a moist chamber. The most aggressive isolate was CMM-1068.

Diversity of corticolous myxomycetes in the last glacial refugia of Cryptomeria japonica in Japan

<em>Cryptomeria japonica</em>, commonly known as Japanese cedar, is now widely distributed from glacial refuges to the entire Japanese archipelago, after the last ice age. The bark surface provides a habitat for many corticolous myxomycetes. Although corticolous myxomycetes are known to prefer tree species, the association between myxomycete distribution and host tree (<em>C. japonica</em>) divergence across the refuges has not been investigated. In this study, myxomycete communities in five refuges were assessed and compared with those in 14 peripheral areas. Bark samples were collected from at least 10 trees per site and were subjected to the moist chamber culture method (10 Petri dishes per tree) to examine the myxomycete fruiting bodies strictly. Environmental variables such as geographical location, climate condition, and bark traits (tree size, bark pH, and electric conductivity) were measured. Fruiting bodies appeared in 91% of the cultures, and 32 taxa (31 species and one varie ty) were recorded. Comparison of the communities between refuges and peripheral sites showed six myxomycete species, Arcyria cinerea, <em>Macbrideola argentea</em>, <em>Cribraria minutissima</em>, <em>Clastoderma debaryanum</em>, <em>Physarum viride</em> and <em>Physarum pusillum</em>, were significantly more abundant in the refuges and these communities preserved higher species diversity. By nonmetric multidimensional scaling, the communities in the Pacific side and the Sea of Japan side were ordered based on snow cover depth, in a pattern similar to the phylogenetic distribution of the host tree. Myxomycete groups were identified in the northern region, the Sea of Japan region, and the southern region (including Yakushima Island) of Japan. Thus, the refugial tree populations preserved the myxomycete species diversity on their bark and functioned as an important hotspot for myxomycetes. The distribution of corticolous myxomycetes was associated with the diversification and biogeographical distribution history of their host tree, <em>C. japonica</em>.

Alternaria tenuissima Causing Leaf Necrosis on Polygonatum cyrtonema in Jinzhai, China

Polygonatum cyrtonema Hua is one of the most important traditional Chinese medicine herbs used for treating stomach weakness, palpitation, diabetes, and dry cough and is commonly planted in the mountain area of southeast and southwest China. During June of 2018 and 2019, necrotic lesions on the leaf apex and leaf margin of P. cyrtonema were observed in seven greenhouses, affecting approximately 2 ha, in Jinzhai County, Anhui Province, China. Disease incidence averaged approximately 30% among all affected greenhouses. The symptoms initially appeared on the leaf apex and leaf margin as small dark brown spots surrounded by light yellow halos. Over time, the lesions enlarged and coalesced to form large areas of necrotic lesions on leaves. To identify the pathogen, 15 symptomatic leaves were collected and surface disinfested with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s, rinsed in sterile water, and dried on filter paper. Plant tissues were then transferred onto potato dextrose agar (PDA) and incubated at 28°C for 3 days in the dark. Five fungal isolates were obtained from the diseased P. cyrtonema leaves by a single-spore isolation method. Colonies of the isolates had round margins and thick fluffy aerial mycelia with olive-green to brown coloration after 5 days on PDA. Conidiophores were pale brown, straight or flexuous, septate, single or in clusters. Conidia were obclavate or oval, multicellular, pale brown, and ranged from 4.91 to 15.83 × 17.29 to 47.32 μm (n = 50), with longitudinal and transverse septa varying from zero to three and from one to six, respectively. Conidia beaks were cylindrical, pale brown, and measured 2.00 to 7.46 × 2.00 to 5.28 μm. On the basis of morphological characteristics, the fungal isolates were preliminary identified as Alternaria spp. (Simmons 2007). To further identify the fungal isolates, the internal transcribed spacer (ITS) region of rDNA of each isolate was amplified and sequenced using the primers ITS4/ITS5 (Guo et al. 2012). The ITS sequence (accession no. MN249629) was 99.45% similar to the reference sequences of Alternaria alternata (KU527784) and A. tenuissima (KX664408) in GenBank, respectively. Partial regions of β-tubulin 2 (TUB2) and histone 3 (H3) genes were sequenced using primers T1/T2 (O’Donnell and Cigelnik 1997) and H3-1a/H3-1b (Glass et al. 1995), respectively. Sequences of TUB2 and H3 (MN256456 and MN256455) of the isolate were 100 and 99.80% similar to the fungus A. tenuissima (JQ811941 and MH647724), respectively. To complete Koch’s postulates, a pathogenicity test of the A. tenuissima isolates was performed on sterile leaves of P. cyrtonema. Ten milliliters of conidial suspension (5 × 10⁵ spores/ml) from each of the five isolates was sprayed on five 6-month-old plants. As a control, five plants were sprayed with sterile water. All treated plants were placed in a moist chamber at 26°C, with 90% relative humidity and an 8-h photoperiod of fluorescent light. Ten days later, 90% of the inoculated plant leaves developed brown spots and necrotic lesions similar to those observed in the field, whereas the control plants were asymptomatic. The same pathogens were again isolated from the necrotic leaves and confirmed as A. tenuissima by DNA sequencing. A. tenuissima is known to cause leaf spot and necrosis on many economically important crops, including lotus (Nelumbo nucifera) and kiwifruit (Actinidia spp.), in China (Li et al. 2019; Zhang et al. 2019). To our knowledge, this is the first report of A. tenuissima causing leaf necrosis on P. cyrtonema in Jinzhai, China.

First Report of Pectobacterium aroidearum Causing Soft Rot in Lettuce and Chinese Cabbage in Brazil

Lettuce (Lactuca sativa L.) and Chinese cabbage (Brassica pekinensis Rupr.) are leafy vegetables of great socioeconomic value in Brazil. Bacterial diseases are among the factors that might limit their yield, particularly soft rot caused by bacteria of the genus Pectobacterium. In July 2014, vegetable gardens of approximately 1.0 and 0.18 ha in the cities of Juazeiro (09° 24′ 42″ S, 40° 29′ 55″ W), state of Bahia, and Petrolina (09° 23′ 55″ S, 40° 30′ 03″ W), state of Pernambuco, Brazil, showed 5% of lettuce plants variety Elba and 44.4% of Chinese cabbage cultivar Michihilli plants with wilt of leaves, foul-smelling soft rot, and inner medulla disintegration. Bacterial isolates were selectively isolated from stem and midribs of lettuce and Chinese cabbage by transferring rot tissue to a healthy pepper fruit using a sterilized toothpick (Takatsu et al. 1981). Pepper fruits were incubated at 26°C for 36 h in a moist chamber, and after incubation bacterial isolates were isolated in Petri dishes containing casamino acid–peptone–glucose (CPG) medium. Small white colonies with “broken glass” aspect (Kelman and Dickey 1995) were observed under a stereoscope, and after that, they were preserved in sterile distilled water (SDW). Twelve isolates were obtained from lettuce and Chinese cabbage plants with soft rot symptoms, which showed the same cultural characteristics in CPG medium. Pathogenicity testing was performed using seedlings of lettuce variety Elba (30 ± 2°C; 62 ± 2% relative humidity [RH]) and detached leaves of Chinese cabbage cultivar Michihilli (28 ± 2°C; 65 ± 2% RH), which are susceptible to bacterial soft rot. Ten microliters of bacterial suspension (10⁶ CFU/ml) were injected into the intermediary portion of the leaf midrib, followed by 48 h of incubation in a moist chamber. For the negative control, seedlings and detached leaves were treated similarly with SDW. The isolate CRMP1 of Pectobacterium aroidearum (Moraes et al. 2017) was used as a reference. Typical symptoms of soft rot were observed 12 to 24 h after inoculation, followed by pathogen reisolation onto CPG medium, fulfilling Koch’s postulates. Seedlings and detached leaves used as negative control remained symptomless, whereas seedlings and detached leaves inoculated with CRMP1 showed soft rot symptoms. Both isolates (UNEB 3 and CCRMPA35) presented gram-negative cells, grew at 37°C, were erythromycin resistant, and were positive for maceration in potato tubers. The Biolog Gen III System identified the isolates as Pectobacterium sp. with a level of 70% similarity. Two isolates were selected for molecular identification, one from lettuce (UNEB3) and one from Chinese cabbage (CCRMPA35). Fragments of the 16S rDNA region (Hauben et al. 1998) and icdA, proA, and mdH genes (Ma et al. 2007) of these isolates were sequenced, and BLASTn analysis of the 16S rRNA region (GenBank MH507334 and MH507330), icdA (GenBank MH500095 and MH500096), proA (GenBank MH507331 and MH507332), and mdH (GenBank MH507329 and MH507330) genes of the isolates UNEB 3 and CCRM35 showed similarity levels of 99, 99, 98, and 99% with sequences of the 16S rRNA region and icdA, proA, and mdH genes of the type isolate of P. aroidearum (isolate SCRI109ᵀ; GenBank JN600323, HM156915, HM157162, and HM156976). Phylogenetic analysis of concatenated sequences of the icdA, proA, and mdH genes using Bayesian inference between isolates UNEB 3 and CCRM35 and species and subspecies of Pectobacterium from GenBank grouped these two isolates with SCRI109ᵀ, with a posterior probability of 100%. To our knowledge, this is the first report of P. aroidearum causing soft rot in lettuce and Chinese cabbage in Brazilian territory. In addition, the present research helps to better know the host range of P. aroidearum in Brazil, which until now was only reported in zucchini (Moraes et al. 2017).

Saccobolus minimus . [Descriptions of Fungi and Bacteria

Abstract A description is provided for Saccobolus minimus , which is often detected only following moist chamber incubation of dung. Some information on its habitat, dispersal and transmission, and conservation status is given, along with details of its geographical distribution (Africa (Libya, Namibia, South Africa), North America (Canada (Ontario, Quebec), USA (Maine, Oregon, Texas, Utah, Vermont)), South America (Argentina, Brazil (Goís, Pernambuco), Chile, Colombia, Ecuador (including Galapagos), Venezuela), Asia (Iraq, Japan, Oman, Taiwan, Thailand), Atlantic Ocean (Ascension Island), Australasia (Australia (Queensland, Western Australia), New Zealand), Caribbean (American Virgin Islands, Dominica, Dominican Republic, Puerto Rico, Saba (Caribbean Netherlands), St Lucia), Europe (Austria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Hungary, Iceland, Italy, Moldova, Netherlands, Poland, Russia (Leningrad Oblast), Serbia, Slovakia, Spain, Sweden, Ukraine, UK), Pacific Ocean (USA (Hawaii))). No evaluation has been made of any possible positive economic impact of this fungus (e.g. as a recycler, as a source of useful products, as a provider of checks and balances within its ecosystem, etc.). Also, no reports of negative economic impacts have been found.

Saccobolus citrinus . [Descriptions of Fungi and Bacteria

Abstract A description is provided for Saccobolus citrinus , which is often detected only following moist chamber incubation of dung. Some information on its habitat, dispersal and transmission, and conservation status is given, along with details of its geographical distribution (Africa (Egypt, Kenya, Tanzania), North America (Canada (Ontario), USA (Oklahoma, Wisconsin)), Central America (Costa Rica), South America (Argentina, Brazil (Goiás, Mato Grosso, Mato Grosso do Sul, Pernambuco, Rio Grande do Sul), Chile, Venezuela), Asia (Armenia, India, Israel, Malaysia, Oman, Taiwan, Thailand), Atlantic Ocean (Portugal (Azores), Spain (Canary Islands)), Australasia (Australia (Queensland, Victoria, Western Australia), New Zealand), Caribbean (Dominica, Guadeloupe, Puerto Rico), Europe (Austria, Belgium, Bulgaria, Czech Republic, Denmark, France, Germany, Greece, Hungary, Iceland, Italy, Netherlands, Poland, Portugal, Russia (Kursk Oblast, Leningrad Oblast), Serbia, Sweden, Switzerland, Ukraine, UK)). No evaluation has been made of any possible positive economic impact of this fungus (e.g. as a recycler, as a source of useful products, as a provider of checks and balances within its ecosystem, etc.). No reports of negative economic impacts have been found.

Proteolytic Activity of The Entomopathogenic Fungi (Penicillium sp. ) of Cockroaches (Periplaneta americana)

Cockroaches (Periplaneta americana) were the insect vectors of disease that caused adverse effects on human health. Control cockroch excessive use of insecticides can lead to residus in the environment and resistance cockroach. Therefore it was necessary to use an alternatives such as such as entomopathogenic fungal as biologycal agents. The entomopathogenic fungi penetrated via the integument of an cockroach to reach the hemocoel. Proteins were the molecules responsible for integument strength in cockroach, It was synthesis the proteases to degrading proteins. The study begins with the isolation of entomopathogenic fungi using the moist chamber method with cockroach as insect bait. Fungus that grow on cockroches are cultured and purifed on Potato Dextrose Agar (PDA) medium then identified. Identification was carried out through macroscopic observations including colony color and diameter and microscopic observations including conidia, conidiophores, hyphae, vesicles, fialids, and leg cells. The result of isolation and identification obtained as Penicillium sp. Proteases enzimatic activity tested on PDA with anlene 1%. The clear zone formed is measured to show the activity of proteases produced by Penicillium sp.

Occurrence of leaf litter inhabiting myxomycetes from lowland forest patches of Northern and Central Vietnam

During the last years, much of the diversity studies of myxomycetes (plasmodial slime molds) have been concentrated mostly in the Southern region of Vietnam. Moreover, information on leaf litter inhabiting myxomycetes for the country is still in scarcity. Hence, this study aims to assess the occurrence and distribution of leaf litter inhabiting myxomycetes in different forest types in the subtropical northern and coastal tropical monsoon central part of the country. Samples of aerial and ground leaf litter that were used to prepare moist chamber cultures in the laboratory were collected in (1) Ba Vi National Park, Ha Noi, (2) Ho Nui Coc, Thai Nguyen, and (3) coastal forest patches in Da Nang. A total of 24 species belonging to 10 genera, wherein the majority of these myxomycete species appeared abundantly (11 species) is reported for this study. Based on species richness, Ha Noi harbored the highest number of myxomycete species. Leaf litter inhabiting myxomycete communities between aerial and ground substrates shared a high level of similarity based on their species composition and relative abundance. Highest level of similarity of leaf litter inhabiting myxomycete asssemblages is also reported between Ha Noi and Da Nang (CC = 0.78, PS = 0.56). This research study is the first step in understanding the complex myxomycete ecology of leaf inhabiting myxomycetes and would help filling now the large gap in one of the unexplored tropical areas of the world.

First Report of Downy Mildew on Blackberry Caused by Peronospora sparsa in Georgia, U.S.A.

HomePlant DiseaseVol. 104, No. 3First Report of Downy Mildew on Blackberry Caused by Peronospora sparsa in Georgia, U.S.A. PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Downy Mildew on Blackberry Caused by Peronospora sparsa in Georgia, U.S.A.M. E. Ali, O. Hudson, S. Waliullah, J. Brock, B. Hayes, J. L. Williams-Woodward, and J. E. OliverM. E. Ali†Corresponding author: M. E. Ali; E-mail Address: ma49268@uga.eduhttp://orcid.org/0000-0002-5871-5055Department of Plant Pathology, University of Georgia, Tifton, GA 31793Search for more papers by this author, O. HudsonDepartment of Plant Pathology, University of Georgia, Tifton, GA 31793Search for more papers by this author, S. WaliullahDepartment of Plant Pathology, University of Georgia, Tifton, GA 31793Search for more papers by this author, J. BrockDepartment of Plant Pathology, University of Georgia, Tifton, GA 31793Search for more papers by this author, B. HayesUniversity of Georgia Cooperative Extension, Mitchell County, Camilla, GA 31730Search for more papers by this author, J. L. Williams-WoodwardDepartment of Plant Pathology, University of Georgia, Athens, GA 30602Search for more papers by this author, and J. E. OliverDepartment of Plant Pathology, University of Georgia, Tifton, GA 31793Search for more papers by this author AffiliationsAuthors and Affiliations M. E. Ali1 † O. Hudson1 S. Waliullah1 J. Brock1 B. Hayes2 J. L. Williams-Woodward3 J. E. Oliver1 1Department of Plant Pathology, University of Georgia, Tifton, GA 31793 2University of Georgia Cooperative Extension, Mitchell County, Camilla, GA 31730 3Department of Plant Pathology, University of Georgia, Athens, GA 30602 Published Online:30 Dec 2019https://doi.org/10.1094/PDIS-09-19-1962-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Blackberries (Rubus L. subgenus Rubus) are small, round, aggregate berries comprising a cluster of tiny fruits called drupelets that grow on flowering shrubs or trailing vines (Clark et al. 2007). The acreage of blackberry production in Georgia has increased 10-fold in the last 15 years to 880 acres (NASS 2017). Downy mildew of blackberry, caused by the oomycete pathogen Peronospora sparsa, is a systemic disease that impacts blackberry production in many regions worldwide. Symptoms of downy mildew on blackberry include yellowing on the upper leaf surface and reddish-brown angular lesions with necrotic centers and chlorotic margins. In May 2019, leaf symptoms typical of blackberry downy mildew were observed on blackberry cultivar ‘Ouachita’ plantlets at a nursery in Mitchell County, Georgia. Initial identification was done based on the morphological characteristics of the sporangia and sporangiophores. Round or slightly ovoid sporangia were observed that were colorless to yellowish-brown, consistent with Peronospora sp. (Gubler and Rebollar-Alviter 2017). The identification of P. sparsa was further confirmed using nucleic acid-based molecular methods directly on infected plant tissue. Genomic DNA was extracted from symptomatic leaf tissue and tested for the presence of P. sparsa by targeting the P. sparsa-specific rDNA-ITS region using “P” primer pair P1 (5′-CACGTGAACCGTATCAACC-3′) and P2 (5′-GATAGGGCTTGCCCAGTAG-3′) with an amplicon size of 94 bp (Hukkanen et al. 2006). PCR reactions produced the expected sizes of the fragments for P. sparsa. The amplified products were then sequenced and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequence of P. sparsa-specific rDNA-ITS (GenBank submission no. MN477916) shared 100% identity to corresponding sequences from P. sparsa (accession no. Y15816). Samples were also tested for the presence of P. sparsa by real-time PCR using the same set of “P” primers. For real-time PCR, plant internal control primer pair R1 (5′-CAAACGACTCTCGGCAAC-3′) and R2 (5′-CCTCGGCCTAATGGCTT-3′) was used to target the highly conserved region of 5.8S plant ribosomal DNA (Hukkanen et al. 2006). The real-time PCR assay was performed in a Cepheid smart cycler II (Sunnyvale, CA) using iQ SYBR Green Supermix (BioRad Laboratories, Hercules, CA) in a 25-µl reaction according to the manufacturer’s protocol (Harper et al. 2010). All four samples extracted from symptomatic leaves were positive based on the real-time PCR assay. In addition, Koch’s postulates were fulfilled to verify the pathogenicity of the P. sparsa. Leaves from four uninfected blackberry plants (also cv. Ouachita) were surface sterilized, laid under sporulating leaf tissue, and incubated at 17°C with a 12-h photoperiod in a moist chamber. After 16 days, on the previously uninfected leaves, sporangiophores and sporangia consistent with P. sparsa developed. As a control, additional leaves from the same four plants were surface sterilized, placed in a moist chamber, incubated at 17°C with a 12-h photoperiod, and examined 16 days later. No lesions or sporulation developed on the uninoculated controls. To the best of our knowledge, this is the first report of P. sparsa causing downy mildew on blackberry in Georgia. Our findings suggest that Georgia blackberry growers should be aware of this disease and accordingly establish management measures for P. sparsa.The author(s) declare no conflict of interest.

Coffee Dieback Caused by Lasiodiplodia pseudotheobromae in Brazil

Coffea arabica is the most important coffee species cultivated worldwide. Although many diseases are known to affect coffee plantations, few pathogens have been reported to cause the death of plants. In 2019, in two growing areas (0.3 and 1 ha) of C. arabica cv. Catuai 144, in Brejao, Pernambuco, Brazil (8°57′28″S; 36°32′00″W; altitude 750 m), 2% of 3-year-old plants showed dieback symptoms. Infected stems had necrotic lesions and black pycnidia on the surface. Three single-spore cultures were obtained directly from three infected stems and deposited in the Culture Collection of Phytopathogenic Fungi Prof. Maria Menezes (CMM) at the Universidade Federal Rural de Pernambuco (Recife, Pernambuco, Brazil) (codes CMM4875 to CMM4877). The colony grown on potato dextrose agar (PDA) medium showed fast growth, with abundant aerial mycelium that was initially white and became grayish. Conidia were initially hyaline, unicellular, and ellipsoidal, 21.4 to 30.7 × 11.4 to 15.1 µm (n = 30). Mature conidia showed a dark brown pigmentation, with a central septum and longitudinal striations. Morphological characteristics were compared with previous descriptions (Alves et al. 2008; Phillips et al. 2013), and the isolates were identified as Lasiodiplodia sp. To complete the identification to species level, sequences of the region of ribosomal DNA (ITS-rDNA) and translation elongation factor 1-α (TEF1-α) were successfully amplified and sequenced, and consensus sequences were deposited in GenBank database (accession nos. MN122134 to MN122136 for ITS and MN103350 to MN103352 for TEF1-α). The BLASTn search of ITS-rDNA and TEF1-α sequences showed 100 and 99% identity, respectively, with reference sequences of the type strain Lasiodiplodia pseudotheobromae CBS116459 (accession nos. EF622077 and EF622057, respectively). A combined phylogenetic tree obtained by Bayesian inference, using MrBayes version 3.1.1 (Ronquist and Huelsenbeck 2003), grouped the isolates within the L. pseudotheobromae clade, confirming the identification. Therefore, based on morphological and molecular analyses, the fungal pathogen was identified as L. pseudotheobromae. The pathogenicity of the three isolates was evaluated in five plants (6 months old) and in 15 detached leaves of C. arabica cv. Catuai 144. Unwounded and superficially wounded (with a sterilized needle) stems and leaves were inoculated by depositing mycelial plugs (5 mm) of 3-day-old colonies grown on PDA medium. As a control, PDA plugs were used. Inoculated plants were maintained in a moist chamber for 2 days and subsequently transferred to a greenhouse under a 12-h photoperiod at 25°C. Inoculated leaves were maintained in a moist chamber at 25°C for 10 days. Only wounded plants and leaves showed symptoms, whereas unwounded and control plants and leaves remained healthy. Necrotic lesions were observed in the plants at 48 h after inoculation (hai), which advanced on the stem and leaves, leading to the death of the top at 10 days after inoculation (dai). Detached leaves showed necrotic lesions at 24 hai, and most tissues were necrotized at 5 dai. Black pycnidia were found on necrotic tissues at 10 dai. The fungus was successfully recovered from inoculated symptomatic tissues, confirming Koch’s postulates. Although L. pseudotheobromae has been reported in C. arabica in Thailand, its pathogenicity has not been confirmed (Trakunyingcharoen et al. 2015). To our knowledge, this is the first report of this species causing dieback on C. arabica in Brazil and in the world. Thus, this finding highlights the occurrence of a new pathogen that can cause losses in coffee cultivation and may contribute to future studies involving disease management in this crop.

First Report of a Leaf Spot Disease Caused by Alternaria protenta on the Datura stramonium in Serbia

Datura stramonium L. (jimsonweed) is an invasive and competitive annual weed and a potential reservoir host of pests and pathogens in agricultural fields. During a survey of potato growing fields in the municipality of Maglic, Vojvodina Province, typical Alternaria symptoms on leaves of D. stramonium were observed among the potato plants in September 2016. The incidence of diseased plants ranged from 12 to 70%. The symptoms started as small, round, light brown spots with chlorotic haloes, which gradually enlarged to 0.3 to 2 cm in diameter, becoming irregular and sometimes having a dark brown border. Isolates were obtained by placing small pieces of surface-sterilized infected tissues (immersed in 1% sodium hypochlorite for 1 min and rinsed with sterilized water) on V8 medium for 7 days at 23°C with a 12-h photoperiod. The five isolated colonies were circular, smooth, gray to brownish black, with concentric zones of very intensive sporulation. Conidiophores were branched, solitary or in small groups, brown to olivaceous brown, ranging from 125 to 230 µm. Mature conidia were olivaceous brown, long ovoid or long ellipsoid, 102 to 113 µm long and 16 to 29 µm wide, with five to 12 transverse septa and zero to three longisepta. Beak dimensions were 62 to 98 µm long and 6 µm wide at the base apex to 2 µm on the top of the beak. Two-beak conidia were noticed with a frequency of 28%. Based on morphological characteristics, all isolates were identified as Alternaria protenta Sorauer (Simmons 2007). To further identify the pathogen, PCR was used with the A. solani-specific primer set OAsF7 and OAsR6 (Gannibal et al. 2014). A single unique band of 164 bp was amplified for all five isolates. To confirm the pathogen’s identity, PCR was carried out with isolate JB 46-1 using primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), and RPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013). The ITS, GAPDH, and RPB2 sequences were deposited in GenBank under accession numbers KY913819, KY913818, and MH454660, respectively, and BLAST analyses showed 100% nucleotide sequence identity with the corresponding gene regions of A. protenta strain CBS 116651 (KC584217, KC584139, and KC584430). A pathogenicity test was carried out with isolates JB 46-1 to JB 46-5 on 20 5-week-old healthy jimsonweed plants grown in a greenhouse at 23 ± 2°C. Inoculation was performed by spraying a conidial suspension (10⁶ spores/ml) prepared from 10-day-old cultures grown on V8 agar plates at 23°C with a 12-h photoperiod. Plants used as controls were sprayed with sterilized distilled water in a similar manner. Inoculated plants were kept in a moist chamber for 2 days with 90 to 100% relative humidity. Leaf spot symptoms, brown lesions with chlorotic halos, similar to those previously observed in naturally infected plants, appeared 5 days postinoculation for all inoculated plants. No symptoms were observed on the inoculated control plants. To fulfill Koch’s postulates, lesions were excised, surface sterilized, and plated on water agar, and the fungus emanating from the infected tissues was identified as previously described. To our knowledge, this is the first report of A. protenta causing leaf spot on D. stramonium in Serbia. According to previous reports, A. protenta can also cause leaf spot disease on potato (Ayad et al. 2017; Landschoot et al. 2017; Woudenberg et al. 2014). The fact that jimsonweed coexists in close proximity with potato plants may have a potentially important economic impact on cultivation of solanaceous crops if left uncontrolled and unmonitored. Therefore, constant management practices are required to control the threat that D. stramonium weed and its diseases might present.

Diversity of <em>Myxomycetes</em> in arid zones of Peru part II: the cactus belt and transition zones

The results obtained from a second survey for Myxomycetes in the arid areas of Peru are reported. A total of 37 localities from the cactus belt (‘cardonal’), between 1500 and 3000 m a.s.l., were sampled over six years. This survey is based on 601 identifiable collections of myxomycetes, developed in the field under natural conditions or those that were recovered from moist chamber cultures. In total, 84 taxa representing 19 genera were recorded. One new species, Cribraria spinispora Lado & D.Wrigley sp. nov., is described, three species new to South America and 22 additional new species for Peru are reported as well. Endemic plants, expecially cacti, had a particular relevance as myxomycete substrates. The predominance of the order Physarales T.Macbr. in arid areas is reinforced. Comments are included on some collections, as well as SEM micrographs of several species. A statistical evaluation of the diversity of myxomycetes in the cardonal area and a comparison of this area with the dry coastal desert of Peru and other Neotropical xeric environments are also included. Results show that the arid areas are rich in myxomycetes, and that each area has a unique assemblage. The differences appear to be related to the host plants.

Infection of the Native California Grass, Bromus carinatus, by Fusarium circinatum, the Cause of Pitch Canker in Pines.

At Point Reyes National Seashore in California, Fusarium circinatum, the causal agent of pitch canker in pines, was isolated from Pinus muricata, the California native grass, Bromus carinatus, and the introduced grass, Holcus lanatus. All grass plants from which F. circinatum was isolated were symptomless. Pathogenicity of grass isolates was confirmed by inoculation of P. radiata trees, which developed symptoms similar to trees inoculated with a pine isolate of F. circinatum. Isolates from grasses were somatically compatible with isolates recovered from symptomatic pines. B. carinatus grown in a growth chamber was inoculated with a green fluorescent protein-expressing strain of F. circinatum. Segments of inoculated leaves were incubated in moist chambers; after 1 to 2 days, sporulating hyphae were observed growing from leaf tissue. Spores of F. circinatum removed from B. carinatus leaves were confirmed to be fluorescent when illuminated with ultraviolet light. These results raise the possibility that B. carinatus cryptically infected by F. circinatum may be a source of propagules capable of infecting pines.

Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic

This study aimed to detect potential bacteria and yeast for prevention of Sclerotinia sclerotiorum infection via biocontrol in garlic. Two yeasts (Pichia kudriavzeviiand Candida labiduridarum) and four bacteria (Bacillus acidiceler, B. macauenses, B. amyloliquefaciens and B. pumilus) were tested. The effect of volatile and diffusible antifungal metabolites on S. sclerotiorum mycelial growth in vitro was evaluated. Garlic cloves were immersed in a suspension of each microorganism (1 × 108 cells ml−1), then the phytopathogen was inoculated and the cloves were kept in a moist chamber for 15 days, at which point lesion diameter was evaluated and electron micrographs were obtained. The results showed a higher percentage of inhibition of S. sclerotiorum growth by volatile metabolites produced by C. labiduridarum, B. macauenses, B. amyloliquefaciensand B. pumilus than by those produced by the other agents, with variation of 74.61%–87.61%. A high reduction in phytopathogen growth due to B. amyloliquefaciens(84%) was observed, suggesting that B. amyloliquefaciens produces antifungal metabolites that inhibit phytopathogen development. The reduction in disease-affected area was most significant in garlic cloves treated with B. pumilus (86.74%) and C. labiduridarum (61.47%). Electron micrographs showed garlic clove surface colonization by all tested microorganisms and phytopathogen hyphae colonization. Research on B. amyloliquefaciens, B. pumilus and C. labiduridarum biocontrol of S. sclerotiorum in garlic is of interest.

Alternaria tenuissima Causing Fruit Scab Disease on Actinidia chinensis in Anhui Province, China

Chinese gooseberry (Actinidia chinensis Planch) is an economically and nutritionally important fruit widely cultivated in mountainous areas of Anhui, Zhejiang, Fujian, Hubei, Hunan, Sichuan, Guizhou, and Shanxi Provinces in China. During 2018 to 2019, a fruit scab disease was identified on the Chinese cultivar Hongyang (A. chinensis var. rufopulpa) in Hefei city, Anhui Province. Irregular, cracked scabs 1 to 5 cm in diameter appeared on approximately 80% of kiwifruits at the survey site. The disease symptoms began as small, pale brown spots on fruits in late May. The spots multiplied and coalesced to form large brown, cracked scabs on immature fruits under high humidity (RH >90%). The flesh beneath the hardened scab was undamaged and green in color. To understand the cause of this disease, 15 symptomatic kiwifruits were cut into small segments and surface sterilized in 70% ethanol for 30 s followed by 0.1% HgCl for 30 s and then washed with deionized water. Pieces of scab tissues were then incubated on potato dextrose agar at 28°C in the dark. After 3 days, four fungal isolates were obtained from the fruit scab tissues by single-spore isolation. Pure cultures formed olive-colored mycelia with a light-colored, taupe-white center. Conidiophores were short, pale brown, septate, arising singly, and measured 21.6 to 58.7 × 2.8 to 4.3 μm (n = 48). Conidia were multiseptate, pale brown, obclavate or obpyriform, with zero to three longitudinal septa and one to four transverse septa, measuring 13.4 to 24.3 × 4.2 to 12.1 µm (n = 48). The pathogen was preliminary identified as Alternaria tenuissima based on morphological characterization (Simmons 2007). To confirm the identification, the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS4/ITS5 (Guo et al. 2012), and the resulting sequence was deposited in GenBank (accession no. MN396735). A BLASTN search showed 99.6% similarity with A. tenuissima (MG214860 and five other accessions; query cover, 100%; E value, 0) and A. alternata (KU324787, query cover, 100%; E value, 0). The ITS sequence did not distinguish A. tenuissima from the A. alternata complex. Therefore, a partial region of the histone 3 (H3) gene was amplified with primers H3-1a/H3-1b (Glass et al. 1995), and the resulting sequence (accession no. MN395834) showed 99.8% similarity with A. tenuissima (MF070841 and 66 other accessions) in the NCBI database. Therefore, this pathogen was identified as A. tenuissima on the basis of morphology and similarity of the H3 gene. To test pathogenicity of the four isolates, 5 ml of conidial suspensions (1 × 10⁶ conidia/ml) of each isolate was sprayed on 10 healthy young fruits of A. chinensis var. rufopulpa. An equal amount of control fruits was inoculated with sterile water. All inoculated fruits were maintained in a moist chamber (RH >90%) at 26°C for 2 weeks. Brown scabs consistent with those observed in the field formed on 60% of the inoculated fruits after 10 days, whereas the controls were asymptomatic. A. tenuissima was reisolated from the diseased fruits, fulfilling Koch’s postulates. The pathogenicity experiment was repeated three times with the same results. A. tenuissima is a known pathogen in China, causing brown leaf spot on kiwifruit (Li et al. 2019). To our knowledge, however, this is the first time A. tenuissima has been shown to cause scabs on the fruit of A. chinensis in Anhui Province, China. This new disease seriously affects the quality and yield of A. chinensis; thus, effective control strategies are urgently needed.

A new iridescent corticolous myxomycete species (Licea: Liceaceae: Liceales) and crystals on American elm tree bark in Texas, U.S.A.

A Licea species new to science is described on the bark surface of living American elm (Ulmus americana L.) cultured in moist chambers. It is characterized by an iridescent peridium on the sides of the sporangium, a black apical circular patch of globular debris, and dark reddish black spores that are smooth over half the surface and ridged-reticulate over the other half with the paler thinner wall collapsing into a coffee-bean shape. This combination of morphological characters is distinct and separates this taxon from all other species of Licea. The history of moist chamber culture use and field collection of corticolous myxomycetes is reviewed. The discovery of crystals of unknown origin on the bark surface of American elms associated with Licea species are illustrated with light microscope photography and scanning electron microscopy. Light microscope images and habit photographs were made using multi-focus imaging and computer stacking to increase depth of field and provide illustrations in color of sporangial structures of the new Licea species. This tiny short-stalked Licea approximately 100 um in height, and with distinctive external and internal morphological characters, was photographed using scanning electron microscopy. Dark-spored versus light-spored species of Licea are reviewed and compared with the most recent molecular analysis as this relates to the genus Licea. This paper is the first in a series that will document the discovery of Licea fruiting bodies of four new species on American elms in nature parks near Fort Worth, Texas.

Isolasi dan Aplikasi Fungi Entomopatogen dari Larva Nyamuk Aedes aegypti L.

DHF (Dengue Hemorrhagic Fever) is a serious problem in Indonesia. DHF disease control has been applied so far, one of which is the use of larvacide temephos (abate). However larvacide is a chemical insecticide that has a negative impact on human health and causes resistance. Therefore in this research, biological control is carried out by utilizing entomopathogenic fungi as a larvacide. The purpose of this study was to determine the effectiveness of entomopathogenic fungi isolated from Ae aegypti larvae. Against the death of Ae. Aegypti mosquito larvae with the moist chamber method. This study used a completely randomized design (CRD) with a factorial pattern and performed two repetitions. Factor A is a type of fungi with 3 levels, namely A1: Aspergillus sp1, A2: Aspergillus sp2, and A3: Syncephalastrum sp. Factor B is a dilution with 7 levels, namely B0: Control, B1: 100 (without dilution), B2: 10-1, B3: 10-2, B4: 10-3, B5: 10-4, B6: 10-5 with every treatment was applied in 2 repetitions. Observations were made 24 hours after treatment for 3 days. Data were analyzed using analysis of variances (anova) and continued with the Least Significant Difference Test (LSD) at 5%. The results indicate that fungi isolates are the most effective in killing Ae. Aegypti mosquito larvae is Aspergillus sp1 and Aspergillus sp2 on the treatment of spores without dilution. 
 
 Key words: DHF; larvacide; Aedes aegypti; Entomopathogenic Fungi.

Investigation of Myxomycetes in Selcen Mountain (Turkey) and its close environs

This study has been made on the specimens which were obtained from 11 different station areas of Selcen Mountain and its close environs in 2011-2012. The samples were acquired from barks of trees, leaves and the materials of decayed trees. These materials were employed the Moist Chamber Culture and it was tried to develop myxomycetes sporophore. In addition myxomycetes were obtained from natural environment. As a result of field and laboratory studies 57 taxa belonging to 10 families and 21 genera were identified, and they were added to the Turkish Myxobiota.