Mechanisms Involved in Biological Control of Dutch Elm Disease

An effective suppression of Dutch elm disease symptom development was observed in elms resistant to the non-aggressive strain of Ophiostoma ulmi, but not to the aggressive strain, after a preventive treatment by this non-aggressive strain. Anequally effective isolate of Verticillium dabliae suppressed symptom development even in the very susceptible field elm (Ulmus carpinifolia). Induction of resistance is proposed for the major mechanism explaining these and earlier experiments. The primary stimuli initiating a resistance reaction are unknown, but the intermediates of the univalent reduction of oxygen may play a role. In an experiment the activities of two groups of enzymes involved in the catalytical scavenging of these radicals, superoxide dismutase and peroxidase, were monitored, but no correlation with induction of resistance was observed. Resistant elms showed the highest activities, probably reflecting a more effective defence against radicals than susceptible ones.     

Resistance of temperate Chinese elms (Ulmus spp.) to feeding by the adult elm leaf beetle (Coleoptera: Chrysomelidae)

Newly acquired Ulmus species from the temperate regions of China growing at The Morton Arboretum, Lisle, IL, were evaluated in laboratory bioassays for feeding preference and suitability for larvae and adults of the elm leaf beetle, Pyrrhalta luteola (Müller). Larval and adult no-choice and adult multiple-choice feeding studies revealed that U changii, U. lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis were the least suitable for feeding and reproduction by the elm leaf beetle. Ulmus wallichiana and the highly preferred U pumila were more suitable for feeding and reproduction. Suitability had no significant effect on male and female longevity. The least suitable species of U. changii, U. lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis show promise for future elm breeding programs.     

Rapid Evolutionary Changes in a Globally Invading Fungal Pathogen (Dutch Elm Disease)

Two enormously destructive pandemics of Dutch elm disease occurred in the 20th century, resulting in the death of a majority of mature elms across much of the northern hemisphere. The first pandemic, caused by Ophiostoma ulmi, occurred as this pathogen spread across Europe, North America and Southwest and Central Asia during the 1920s–1940s. The current pandemic is caused by another Ophiostoma species, O. novo-ulmi. Since the 1940s, O. novo-ulmi has been spreading into the regions previously affected by O. ulmi. It has also spread as two distinct subspecies, termed subsp. americana and subsp. novo-ulmi. This sequence of events has resulted in competitive interactions between these previously geographically isolated pathogens. This article summarizes the biological properties of the Dutch elm disease pathogens and their history of spread. It reviews the remarkable series of genetic events that have occurred during their migrations; including the emergence of genetic clones, the spread of deleterious fungal viruses within the pathogen clones, and the rapid and continuing evolution of O. novo-ulmi via horizontal gene flow. The wider role of horizontal gene flow in the evolutionary potential of migratory plant pathogens is discussed.     

Ecology and diversity of waxcap (Hygrocybe spp.) Fungi

Summary

Members of the genus Hygrocybe are ubiquitous and colourful components of many undisturbed and nutrient-poor grasslands in the UK. Through a number of detailed surveys of the distribution of Hygrocybe spp. and of genera showing similar patterns of occurrence (e.g. Clavaria spp., Entoloma spp., Geoglossum spp.) a picture is gradually emerging of the more important ‘waxcap grassland’ sites, and of those species in greatest need of protection. Waxcap fungi are far from ideal experimental organisms which explains why so little has been published about their biology and ecology. They cannot be cultured on laboratory media and the correct conditions for inducing spores of most species to germinate have yet to be established. Nevertheless approaches such as isotope ratio mass spectrometry and the use of molecular biology techniques are beginning to provide an insight into the role played by these organisms in grassland ecosystems, and why they are so adversely affected by many agricultural practices. Current field experiments at various sites including Sourhope near Kelso will also permit investigations into waxcap ecology to be correlated with parallel studies of other members of the soil biota.     

Resistance of Enterobacter sakazakii (Cronobacter spp.) to environmental stresses

Aim:  To gain a better understanding of the survival and persistence of Enterobacter sakazakii in severe environments.
Methods and Results:  We evaluated the resistance of Ent. sakazakii to various environmental stresses, including heating, drying, water activity ( a _w), and pH. The resistance of Ent. sakazakii to heat varies widely among strains. Most tested strains of Ent. sakazakii exhibited unusual resistance to dry stress, which depends on drying media. Growth of most strains occurred within 24 h at 37°C when the initial a _w of the medium was adjusted to 0·94 with sucrose or sodium chloride. The minimum pH for growth within 24 h at 37°C was 3·9 or 4·1 for most strains tested. Additionally, there did not appear to be any relationship between resistance to stresses and biofilm-forming ability in Ent. sakazakii planktonic cells.
Conclusions:  These results indicate that Ent. sakazakii is much more resistant than other Enterobacteriaceae to environmental stresses. Moreover, it is likely that Ent. sakazakii has cross-resistance to dry and thermal stresses.
Significance and Impact of the Study:  The findings of this study will contribute to an improved understanding of the survival and behaviour of Ent. sakazakii , which will lead to improved strategies for preventing outbreaks of Ent. sakazakii infection.     

Resistance to broomrape (Orobanche spp.) in sunflower (Helianthus annuus L.) is temperature dependent

The effects of various temperature regimes in the range 29-17/21-9 degrees C day/night on each stage of the parasitism process of Orobanche cumana and O. aegyptiaca on sunflower were studied under controlled conditions in polyethylene bags. The response of the resistant sunflower variety 'Ambar' was expressed as the degeneration of the parasite tissues after its establishment in the plant roots, and this stage was found to be temperature dependent. The degeneration rate of Orobanche tubercles in the resistant sunflower variety was also found to be temperature dependent and was about five times as great as that in the sensitive variety in the highest temperature regime tested of 29/21 degrees C day/night. The ability to reject the parasite by causing its degeneration and death is the main factor that determines the resistance. As the temperature rises, more tubercles degenerate and die, that is the sunflower plant expresses higher levels of resistance.     

Endophytic insect communities of two prairie perennials (Asteraceae: Silphium spp.)

Little is known of the biology of most insects that are endemic to prairie ecosystems of North America, with the exception of large and conspicuous species. In particular, species that are sequestered within plant tissues are commonly overlooked. In this paper, we assess the biodiversity of endophytic insects that inhabit stems of Silphium laciniatum L. and S. terebinthinaceum Jacquin (Asteraceae), endemic plants of tallgrass prairies. Endophytic herbivores, gall wasps Antistrophus rufus Gillette and A. minor Gillette (Hymenoptera: Cynipidae) and stem-boring larvae of the beetle Mordellistena aethiops Smith (Coleoptera: Mordellidae) were attacked by 10 species of natural enemies. We report new host plant associations for herbivores, and new host insect associations for parasitoids. The two plant species differed significantly in their densities of gall wasps and the vertical dispersion of galls within stems. Interactions within and between trophic levels attest to the biodiversity of endophytic insect communities, and the specialized nature of these insects suggests they are highly vulnerable to habitat conservation practices that involve destruction of dead vegetation.     

Two Endophytic Fungi in Different Tissues of Scots Pine Buds (Pinus sylvestris L.)

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.     

白木香内生真菌的分离鉴定及其抑菌活性

从白木香Aquilaria sinensis（Lour．）Gilg木质部的树脂形成部位和健康部位中共分离获得42株内生真菌,经初步鉴定,产孢的33株分属于3目4科7属,其余未产孢的9株暂归为无孢菌群。采用杯碟法和MTT法分别测定了各菌株的发酵上清液对3种病原菌的体外抑菌活性和2种肿瘤细胞的体外细胞毒活性。结果表明,白木香木质部健康部位内生真菌以枝顶孢霉属为优势属,而树脂形成部位的内生真菌种类比健康部位要多,且以青霉属为优势属。其中26株至少能抑制一种指示菌,占总数的61．9％;7株对指示瘤株具有细胞毒活性,占总数的16．7％。抑菌活性菌株主要分布在枝顶孢霉属和青霉属。枝顶孢霉属菌株抑菌活性较强,其抗菌活性成分值得进一步研究。     

Screening Subterranean Clover (Trifolium spp.) Germplasm for Resistance to Meloidogyne species

This study was conducted to identify lines of subterranean clover (Trifolium spp.) with resistance to Meloidogyne arenaria (Neal, 1989) Chitwood, 1949, race 1; M. incognita (Kofoid and White, 1919) Chitwood, 1949, race 3; and M. javanica (Treub, 1885) Chitwood, 1949. A collection of 134 subterranean clover lines was evaluated and all had intermediate to high susceptibility. Root galling was negatively correlated with both seed and dry matter yields. Soil fumigation significantly reduced the nematode population in the field. Results indicate there is limited genetic resistance to root-knot nematodes among subterranean clover lines. Alternative sources of variation for this trait should be investigated.     

Endophytic Fungi in Four Winter Wheat Cultivars (Triticum aestivum L.) Differing in Resistance Against Stagonospora nodorum (Berk.) Cast. & Germ. =Septoria nodorum (Berk.) Berk.

Four winter wheat cultivars with different levels of resistance to Septoria nodorum were investigated at four locations during two vegetation periods. Forty plants per cultivar and site were collected at random at seven defined growth stages from crop emergence to harvest. Samples from roots, culms, leaves, glumes and kernels were examined for the occurrence of endophytic fungi after surface sterilization. 83% of the 26944 isolates sporulated and were assigned to 213 species. The most frequent were: Septoria nodorum (20.1%), Alternaria tenuissima (9.8%), Epicoccum purpurascens (9.1%), Idriella bolleyi (6.9%), Fusarium graminearum (5.3%), Fusarium culmorum (4.0%), Cladosporium oxysporum (3.7%), Didymella exitialis (3.1%), Fusarium nivale (2.8%) and Rhizoctonia solani (2.1%). Each species occurred preferentially in one or more plant organs. A factorial analysis of variance showed that plant organ, sampling site, vegetation, period and cultivar in decreasing order of importance influenced the quantitative and qualitative composition of the fungal populations. No relationship between endophytic fungi was found to be constantly antagonistic or mutualistic. Septoria nodorum was isolated mainly from culms. The number of S. nodorum isolates differed significantly between cultivars in culms and glumes but not in flag leaves. The results are discussed in relation to resistance breeding and the effect endophytic fungi, might have on yield.     

Preference of temperate Chinese elms (Ulmus spp.) for the adult Japanese beetle (Coleoptera: Scarabaeidae)

Trees of newly acquired Ulmus species originating from the temperate regions of China and growing at The Morton Arboretum, Lisle, IL, were evaluated in no-choice and multiple-choice laboratory bioassays and cage-studies for feeding preference for adults of the Japanese beetle, Popillia japonica Newman. Ulmus lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis were less preferred for feeding by adult Japanese beetles. No-choice, multiple-choice, and cage feeding studies revealed that Ulmus wallichiana was moderately preferred, and U. procera and U. americana were highly preferred for feeding. Dry fecal pellet weights were strongly correlated with the percent of leaf tissue removed. Feeding preference in the multiple-choice study was strongly correlated with feeding preference in the cage study and in the no-choice study. Likewise, adult feeding preference in the cage study was strongly correlated with preference in the multiple-choice and no-choice studies. The less preferred species of U. lanceafolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis show promise for future elm breeding programs.     

Fungi associated with storage rots of cocoyams (Colocasia spp.) in Nsukka,Nigeria

Cocoyam (Colocasia spp.) corms and cormels showing spoilage symptoms were collected from many stores in Nsukka locality and examined for rot and associated fungal pathogens. Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum, and F. solani were recovered from rotten cocoyams. The representative isolates of these species caused cocoyam rot in pathogenicity tests. The rot due to A. niger, B. theobromae and C. rolfsii was extensive resulting in complete maceration of cocoyam tissue. Potassium sorbate (0.1 mg/ml) protected cocoyams from fungal rot with the exception of C. rolfsii.     

Anatomical and nutritional factors associated with susceptibility of elms (Ulmus spp.) to the elm leaf beetle (Coleoptera: Chrysomelidae)

A wide range of susceptibility exists across elm (Ulmus) species and hybrids to the elm leaf beetle, Pyrrhalta luteola (Müller) (Coleoptera: Chrysomelidae). We evaluated various elm species, hybrids, or cultivars (taxa) growing in an experimental plantation in the city of Holbrook, AZ, for leaf anatomical (toughness and trichome density) and nutritional (minerals and sugars) traits that may be associated with host resistance. Leaf toughness and percentage of defoliation (susceptibility) were not correlated. However, we found weak negative correlations between percentage of defoliation and density of trichomes on the leaf abaxial surface. Of the 11 leaf nutrients examined, concentrations of iron and phosphorus correlated inversely with percentage of defoliation. The remaining nine traits did not show any correlation with percentage of defoliation. We concluded that individual anatomical and nutritional traits of elm species/hybrids do not seem to create a strong barrier to elm leaf beetle defoliation. However, the results from a stepwise multiple regression analysis indicated that collectively, these traits may play an important role in determining susceptibility.     

野葛内生真菌的分离、鉴定及体外细胞毒活性

目的:药用植物内生真菌是一类重要的微生物资源,其代谢产物有着广泛的生物学活性。该研究拟从野葛(Pueraria lobata(Willd.)Ohwi)中分离有细胞毒活性的内生真菌。方法:组织块改良法分离内生真菌;细胞毒活性测定采用Alamar blue法;结合形态学和分子生物学方法进行菌种鉴定。结果:获得的34株内生真菌中菌株KLBMP f0027对HepG2、HO-8910、NCI-H460、SGC-7901等细胞株均有显著活性,ID50分别为437.4、460.0、542.5、771.2。而且活性产物相对稳定,对温度、pH变化及蛋白酶不敏感。代谢过程研究显示菌株KLBMP f0027的活性产物合成与细胞生长属于部分耦联关系类型。形态学和ITS-rDNA序列分析鉴定菌株为Aspergillus ostianus strain KLBMP f0027。结论:野葛内生真菌A.ostianus strain KLBMP f0027可作为细胞毒活性候选菌株进行深入研究。     

Susceptibility of 32 elm species and hybrids (Ulmus spp.) to the elm leaf beetle (Coleoptera: Chrysomelidae) under field conditions in Arizona

We evaluated elm leaf beetle, Pyrhalta luteola (Müller) (Coleoptera: Chrysomelidae), defoliation of 32 elm species or hybrids (taxa) established under field conditions in Holbrook, AZ. Percentage of defoliation, number of eggs, and number of larvae were estimated on randomly selected 15-cm shoot lengths annually in July, from 1996 to 2001. The following nine taxa consistently sustained 15-46% mean overall defoliation: 1) Siberian elm, U. pumila L.; 2) 'Dropmore' elm, U. pumila; 3) 'Camperdownii' elm, U. glabra Huds.; 4) 'Regal' elm, U. glabra x U. carpinifolia Gledisch x U. pumila); 5) 'Sapporo Autumn Gold' elm (U. pumila x U. japonica Sang.); 6) 'New Horizon' elm (U. pumila x U. japonica); 7) 'Charisma' elm [(U. japonica x U. wilsoniana Schneid.) x (U. japonica x U. pumila)]; 8) 'W2115-1' elm (U. parvifolia Jacq. x U. procera Salisb.); and 9) 'Homestead' elm [(U. hollandica Mill. x U. carpinifolia) x (U. pumila-racemosa Dieck x U. carpinifolia)]. Percentage of defoliation was significantly low on four Chinese elm (U. parvifolia) cultivars ('Allee', 'Athena', 'Glory'/lace bark, and 'Kings Choice'). Percentage of defoliation was also low on seven Asian elms (including U. chemnoui Cheng, U. bergmaniana Sneid., U. szechuanica Fang, and species of the U. davidiana Planch. complex [U. davidiana, U. japonica, U. wilsoniana, and U. propinqua Koidz.]) and the American elm (U. americana L.) 'Valley Forge'. Percentage of defoliation and the number of eggs or larvae per plant were highly correlated. The results of this study are generally consistent with results of past laboratory screening trials.     

药用植物二色补血草内生真菌分离及其抑菌活性初步研究

对药用植物二色补血草内生真菌进行分离,以番茄灰霉病菌、黄瓜枯萎病菌、枸杞黑果病菌、黄瓜立枯病菌、小麦全蚀病菌、枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌和铜绿假单胞菌为靶标菌,采用对峙法和改进的菌块法进行抗菌活性初步筛选。结果表明:(1)从二色补血草根、茎、叶组织器官中分离出18株内生真菌,其中以叶部最多,根部和茎部次之;经形态学初步鉴定归于2目,2科,4属。(2)有10株内生真菌对2种或多种植物病原真菌有不同程度的抑制作用,占分离菌株总数的55.6%;8株内生真菌对1种或多种供试细菌具有不同程度的抑制作用,占分离菌株总数的44.4%;菌株LBEFL001和LBEFL006分别对5种供试真菌具有明显抑制作用,属于曲霉属和青霉属;菌株LBEFR006、LBEFS002分别对金黄色葡萄球菌和铜绿假单胞菌2种供试细菌具有明显抑制作用,属于梭孢霉属;菌株LBEFL004对枯草芽胞杆菌和金黄色葡萄球菌2种供试细菌具有明显抑制作用,属于曲霉属。研究发现,二色补血草具有较为丰富的内生真菌资源,对植物病原真菌具有明显的抑菌活性,且抑菌范围较宽;对病原细菌抑制作用具有一定的选择性,总体上对金黄色葡萄球菌抑制作用明显。     

A Role of the Gelatinous Matrix in the Resistance of Root-Knot Nematode (Meloidogyne spp.) Eggs to Microorganisms

The survival of eggs of the root-knot nematode Meloidogyne javanica was studied in a series of experiments comparing the infectivity of egg masses (EM) to that of separated eggs (SE). The EM or SE were placed in the centers of pots containing citrus orchard soil and incubated for 24 hours, 10 days, or 20 days. Following each incubation time, 10-day-old tomato plants were planted in each pot, and 3 to 4 weeks later the plants were harvested and the galling indices determined. In the EM treatments, galling indices of ca. 4.0 to 5.0 were recorded after all three incubation periods; in the SE treatments, the infectivity gradually declined to trace amounts by 20 days. Incubating EM and SE for 2 weeks in four different soil types showed the same pattern in all the soil types: EM caused heavy infection of the test plants while the infection rate from the SE was extremely low. Incubating EM and SE in soil disinfested with formaldehyde resulted in comparable galling indices in most treatments. In petri dish experiments, 100 mg of natural soil was spread at the perimeter of a Phytagel surface and EM or SE of M. incognita were placed in the center. Light microscopy revealed that within 5 to 10 days the SE were attacked by a broad spectrum of microorganisms and were obliterated while the eggs within the EM remained intact. Separated eggs placed within sections of gelatinous matrix (GM) were not attacked by the soil microorganisms. When selected microbes were placed on Phytagel surfaces with EM of M. incognita, electron microscopy demonstrated that at least some microbes colonized the GM. As the major difference between the EM and the SE was the presence of the GM, the GM may serve as a barrier to the invasion of some microorganisms.     

In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.Key words: Zataria multiflora, antifungal, endophytic fungi, Monosporascus cannonballus

The term “Endophytes” denotes microorganisms that colonize plants’ internal tissues for part of or throughout their life cycle without producing any apparent adverse effect. The endophytic microorganisms include fungi, bacteria, and actinobacteria (Bacon and White 2000). Among them, fungi are the most common endophytic microorganisms (Staniek et al. 2008). Endophytic fungi are ecologically distinct polyphyletic groups of microorganisms, mostly belonging to the Ascomycota phylum (Jia et al. 2016). Several fungal endophytes have been shown to act as biological control agents for managing soil-borne plant pathogens (Toghueo et al. 2016).Zataria multiflora Boiss. (Synonyms: Zataria bracteata Boiss.; Zataria multiflora var. elatior Boiss), belonging to the Lamiaceae family is a traditional medicinal plant commonly used as a flavor ingredient in different types of foods (Sajed et al. 2013). Several medicinal properties of Z. multiflora, including antiseptic, anesthetic, antispasmodic, antioxidant, antibacterial, and immunomodulatory activities, have been documented (Sajed et al. 2013). However, studies on the endophytic microorganisms inhabiting Z. multiflora are limited (Mohammadi et al. 2016).Monosporascus cannonballus Pollack & Uecker (Ascomycota, Sordariomycetes, Diatrypaceae) is one of the most important phytopathogenic fungi causing root rot and vine decline disease in muskmelon. It causes sudden wilt and collapse of the plant at the fruiting stage, which may result in total yield loss (Martyn and Miller 1996). The fungus also infects pumpkin, cucumber, courgette, and watermelon plants (Mertely et al. 1993). The control of M. cannonballus in melon and other cucurbit crops is difficult because of the pathogen’s soil-borne nature. Earlier reports indicated that arbuscular mycorrhizal fungi (AMF) (Aleandri et al. 2015), hypovirulent isolates of M. cannonballus (Batten et al. 2000), Trichoderma spp. (Zhang et al. 1999), and antagonistic rhizobacteria (Al-Daghari et al. 2020) are effective agents for the reduction of M. cannonballus-induced root rot and vine decline of melon. In addition, it is well established that many endophytic fungi isolated from medicinal plants possess antimicrobial activity against phytopathogenic fungi (Jia et al. 2016). The objective of this study was to investigate the presence of endophytic fungi in Z. multiflora and to study theirs in vitro antagonistic activity against M. cannonballus.Z. multiflora plants (accession number 201100114) were obtained from Oman Botanic Garden, Al-Khoud, Sultanate of Oman. The plants were healthy, showing no apparent symptoms of any disease or pest infestation. A virulent isolate of M. cannonballus (ID14367), obtained from the roots of a melon plant showing root rot and vine decline (Al-Rawahi et al. 2018) was used in this study. The culture was maintained on potato dextrose agar (PDA) medium (Oxoid Ltd., Basingstoke, UK).To isolate endophytic fungi, Z. multiflora plants were washed in tap water to remove adhering soil particles. The leaves were separated, cut into small pieces, and surface-sterilized by washing in 70% (v/v) ethanol for 1 min and then in 1% (v/v) sodium hypochlorite for 1 min. The plant tissues were then washed 3–4 times with sterilized distilled water. The leaf tissue pieces were further cut into small pieces (0.2–0.5 cm in length) using a sterile scalpel and placed on PDA medium. The plates were incubated at 25 ± 2°C for 7–10 days, and pure cultures of the endophytic fungi were obtained (Lu et al. 2012).DNA was extracted from the mycelia for molecular identification of endophytic fungi according to the method described by Liu et al. (2000). PCR amplification of the Internal Transcribed Spacer (ITS) regions of the fungal rDNA was performed using the primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) as described by Halo et al. (2018). The PCR products of the expected sizes were sequenced at Macrogen, Seoul, Korea. The sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).A dual culture technique was used to test the in vitro antagonistic effect of the endophytic fungi against M. cannonballus. A mycelial plug (7-mm diameter) was excised from the fungal endophyte colonies and placed on one side of a PDA plate (90-mm diameter) about 1 cm away from the edge. On the same plate, a 7-mm diameter disc of M. cannonballus was placed on the opposite side at 1 cm distance from the edge. The Petri plates inoculated with M. cannonballus alone were used as control. Four Petri plates per treatment were used. The Petri plates were incubated at 25 ± 2°C, and the radial growth of M. cannonballus was measured after 5–7 days of incubation. The mycelial growth inhibition was calculated using the following formula: % inhibition=[1−(T/C)]×100where C – radial growth of M. cannonballus in the control plate and T – radial growth of M. cannonballus in the dual culture plate (Toghueo et al. 2016).To investigate the antagonistic effects of the endophytic fungi on the morphology of M. cannonballus hyphae, the five-mm agar plug samples of M. cannonballus were excised from the colony edges of inhibition zone in the dual culture plate. The samples for scanning electron microscopy were prepared according to the method reported by Goldstein et al. (2003) and observed with a JEOL (Model: JSM-7800F) scanning electron microscope. The culture of M. cannonballus grown in the absence of endophytic fungi served as control.To perform the electrolyte leakage assay, the endophytic fungi were cultured in 200 ml of Czapek Dox broth (static) in 500 ml conical flasks at room temperature (25 ± 2°C) for 14 days, and the culture filtrates were obtained by filtering through Whatman No. 1 filter paper. Five hundred mg of M. cannonballus mycelium were added to 20 ml of culture filtrate in a glass vial. The conductivity of the suspension was measured at 0, 1, and 3 h after incubation by using a conductivity meter (Halo et al. 2018). There were three replicates per treatment and control.Data from the in vitro growth inhibition and the electrolyte leakage assays were statistically analyzed using general linear model ANOVA using Minitab Statistical Software version 17 (Minitab Inc., State College, USA). When ANOVA revealed significant differences between treatments, means were separated using Tukey’s studentized range test at p ≤ 0.05. Arc sine transformation of data on % mycelial growth inhibition was done prior to analysis.A total of five morphologically distinct fungal endophytes were obtained from the leaves of Z. multiflora. Based on the rDNA ITS sequence analysis, these endophytic fungal (Ascomycota, Sordariomycetes) isolates were identified as Nigrospora sphaerica (Amphisphaeriales, Apiosporaceae) (E1 and E6), Subramaniula cristata (Sordariales, Chaetomiaceae) (E7) and Polycephalomyces sinensis (Hypocreales, Ophiocordycipitaceae) (E8 and E10). The sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers {"type":"entrez-nucleotide","attrs":{"text":"MH028052","term_id":"1509265912","term_text":"MH028052"}}MH028052, {"type":"entrez-nucleotide","attrs":{"text":"MH028054","term_id":"1509265914","term_text":"MH028054"}}MH028054, {"type":"entrez-nucleotide","attrs":{"text":"MH028055","term_id":"1509265915","term_text":"MH028055"}}MH028055, {"type":"entrez-nucleotide","attrs":{"text":"MH028056","term_id":"1509265916","term_text":"MH028056"}}MH028056, and {"type":"entrez-nucleotide","attrs":{"text":"MH028058","term_id":"1509265918","term_text":"MH028058"}}MH028058. P. sinensis is an important medicinal fungus. Numerous pharmacological activities of P. sinensis including immunomodulatory, anti-estrogenicity and antitumor activities have been documented (Wang et al. 2012). N. sphaerica has been reported as an endophyte (Wang et al. 2017) as well as a pathogen in a few plant species (Wright et al. 2008; Liu et al. 2016). However, Z. multiflora plants colonized with these endophytic fungi were healthy and did not show any observable disease symptoms.The in vitro dual culture antagonism assay showed that all the five endophytic fungi inhibited the mycelial growth of M. cannonballus. N. sphaerica E1 was the most effective (81.7%), followed by P. sinensis E8 (80.6%), P. sinensis E10 (75.8%) and N. sphaerica E6 (66.1%). S. cristata E7 was the least effective, which recorded 38.7% inhibition (Table ​(TableI,I, Fig. ​Fig.1).1). Further, scanning electron microscopic observations of the hyphae of M. cannonballus from the dual culture assay plates at the edge of the inhibition zone revealed morphological abnormalities such as disintegration, shrinkage, and loss of turgidity. Scanning electron micrograph of M. cannonballus after co-cultivation with the endophytic fungus N. sphaerica E1 is shown in Fig. ​Fig.2.2. These findings corroborate with those of Hajlaoui et al. (1992) who reported plasmolysis of Sphaerotheca pannosa var. rosae mycelium due to the antagonistic effect of Sporothrix flocculosa. Halo et al. (2018) reported shrinkage of Pythium aphanidermatum hyphae due to the antagonistic activity of Aspergillus terreus. The shrinkage of M. cannonballus hyphae in the present study suggests a possible leakage of cytoplasmic contents (Garg et al. 2010). The loss of the turgidity of M. cannonballus hyphae indicates alterations in the permeability of the cell membrane (Halo et al. 2018). Several reports indicate the production of antimicrobial substances by endophytic fungi (Zhao et al. 2012; Homthong et al. 2016). Kim et al. (2001) demonstrated that phomalactone, a compound produced by N. sphaerica restricted the mycelial growth and germination of sporangium and zoospore of Phytophthora infestans and decreased the incidence of late blight in tomato. Zhao et al. (2012) characterized four secondary antifungal metabolites viz., dechlorogriseofulvin, griseofulvin, mullein, and 8-dihydroramulosin from the liquid cultures of the endophytic fungus Nigrospora sp. isolated from roots of the medicinal plant, Moringa oleifera. Homthong et al. (2016) reported the production of chitinase by Paecilomyces (Polycephalomyces) sp. The inhibitory effect of endophytic fungi on the hyphae of M. cannonballus in this study might be due to the production of antifungal metabolites.Table IPercentage inhibition of mycelial growth of M. cannonballus by endophytic fungi isolated from Zataria multiflora in dual cultures on PDA.

PCR-Based Detection of the Causal Agent of Watermark Disease in Willows (Salix spp.)

The watermark disease, caused by Brenneria salicis (formerly Erwinia salicis), is of significant concern wherever tree-forming willows are grown or occur naturally. The movement of infected, asymptomatic cuttings is a major cause of pathogen dispersal. A reliable and sensitive diagnostic procedure is necessary for the safe movement of willow planting material. We derived primers from the nucleotide sequence of the 16S rRNA gene of B. salicis for the development of a PCR to detect this pathogen. One set of primers, Es1a-Es4b, directed the amplification of a 553-bp fragment from B. salicis genomic DNA as well as B. salicis cells. PCR products were not observed when genomic DNA was tested for 27 strains of other, related plant-associated bacteria. Genomic fingerprinting by amplification fragment length polymorphism of B. salicis strains, originating from four different countries, and related Brenneria, Pectobacterium, and Erwinia strains revealed a very high similarity among the B. salicis genomes, indicating that the spread of the pathogen is mainly due to the transportation of infected cuttings. The PCR had to be preceded by a DNA extraction in order to detect the pathogen in the vascular fluid of willows. The minimum number of cells that could be detected from vascular fluid was 20 CFU/ml. The PCR assays proved to be very sensitive and reliable in detecting B. salicis in willow plant material.