Host Range Tests Research Articles

Natural enemies of Lemna minuta in its native range and their potential as biological control agents for Europe

ABSTRACT Lemna minuta is native to North America but an invasive alien plant in Europe, where it poses significant threats to freshwater ecosystems. Explorations for biological control agents were conducted in two regions of the United States (Louisiana and California), revealing three candidate species. Subsequent laboratory investigations focused on the weevil Tanysphyrus lemnae and the fly Lemnaphila scotlandae as the most promising agents and confirmed that both the herbivorous insects attack L. minuta plants. To evaluate the host specificity of these two potential agents, insects were exposed to five species of duckweed commonly occurring in North America and Europe. Preliminary host-range testing indicated that T. lemnae develops on all evaluated duckweed species, including those from different genera (Lemna, Spirodela, Landoltia). Conversely, data revealed that feeding and development of the dipteran L. scotlandae are limited to species in the genus Lemna, but this includes L. minor, a native European species threatened in Europe due to the spread of L. minuta. No-choice and choice tests confirmed that neither larvae nor adults of L. scotlandae discriminate between the two Lemna species, except for pupation, which occurs more frequently in L. minor under choice conditions. We conclude that the broad host-range of T. lemnae and L. scotlandae render them unsuitable as biological control agents of L. minuta in Europe.

An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China.

Chickpea (Cicer arietinum L.) is an important legume crop worldwide. An emerging disease, basal stem rot with obvious wilt symptoms, was observed in the upper part of chickpea plants during the disease survey in Qiubei County of Yunnan Province. Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue of chickpea plants collected from the field. Those isolates were morphologically found to be similar to Diaporthe aspalathi. Molecular sequence analyses of multiple gene regions (ITS, tef1, tub2, cal, and his3) indicated that the three isolates showed a high identity with D. aspalathi. Pathogenicity and host range tests of the isolates were performed on the original host chickpea and eight other legume crops. The isolates were strongly pathogenic to chickpea and appeared highly pathogenic to soybean, cowpea, and mung bean; moderated or mild pathogenic to adzuki bean and common bean; however, the isolates did not cause symptoms on grass pea (Lathyrus sativus). Diaporthe aspalathi was previously reported as a main pathogen causing the southern stem canker in soybean. To our knowledge, this is the first report of D. aspalathi inducing basal stem rot on chickpea worldwide.

Isolation and Characterization of Six Novel Fusobacterium necrophorum Phages.

Fusobacterium necrophorum, a human and animal pathogen, is the primary etiologic agent of bovine liver abscesses and a driving factor for prophylactic antibiotic use in the fed cattle industry. Considering calls to reduce agricultural antibiotic use, we isolated phages capable of killing F. necrophorum as an alternative or complementary biocontrol strategy. Six novel phages (φFN37, φRTG5, φKSUM, φHugo, φPaco, and φBB) were isolated from rumen fluid or ruminal F. necrophorum isolates and subjected to host range testing on both F. necrophorum subspecies. Four F. necrophorum subspecies, necrophorum phages, were tested for cross-resistance and host growth inhibition individually and in pairs. Additionally, genomic sequencing, annotation, and analysis were performed.s. Four of six isolated phages were able to form lysogens, although all six contained lysogeny-related genes. φKSUM and φBB, did not form lysogens and were able to infect both subspecies. Four phages could infect F. necrophorum 8L1 (a liver abscess model challenge strain) in vitro. Genomic analysis showed that these phages belong to class Caudoviricetes with genome sizes ranging from 35 kbp to 111 kbp and GC values ranging from 26% to 36% and have extremely limited similarity to other deposited phage genomes infecting Fusobacterium or other genera. Although all phages isolated contained sequences bearing similarities to genes implicated in lysogeny, the four selected for use in cocktails showed potential in inhibiting host growth, with several demonstrating promising attributes for biocontrol and therapeutic applications. Phage cocktails that may offer enhanced antibacterial activity were also identified, indicating the potential of some lysogenic phages to be adapted for biocontrol or therapeutic purposes when lytic phages are difficult to obtain.

Isolation, characterization, and application of bacteriophage on Vibrio parahaemolyticus biofilm to control seafood contamination

ObjectiveThis study intended to isolate a Vibrio-particular phage from the natural environment, analyse its characteristics and genome sequence, and investigate its reduction effect on V. parahaemolyticus biofilm as a biocontrol agent in squid and mackerel. MethodsAmong 21 phages, phage CAU_VPP01, isolated from beach mud, was chosen for further experiments based on host range and EOP tests. When examining the reduction effect of phage CAU_VPP01 against Vibrio parahaemolyticus biofilms on surfaces (stainless steel [SS] and polyethylene terephthalate [PET]) and food surfaces (squid and mackerel). ResultsThe phage showed the most excellent reduction effect at a multiplicity-of-infection (MOI) 10. Three-dimensional images acquired with confocal laser scanning microscopy (CLSM) analysis were quantified using COMSTAT, which showed that biomass, average thickness, and roughness coefficient decreased when treated with the phage. Colour and texture analysis confirmed that the quality of squid and mackerel was maintained after the phage treatment. Finally, a comparison of gene expression levels determined by qRT-PCR analysis showed that the phage treatment induced a decrease in the gene expression of flaA, vp0962, andluxS, as examples. ConclusionThis study indicated that Vibrio-specific phage CAU_VPP01 effectively controlled V. parahaemolyticus biofilms under various conditions and confirmed that the isolated phage could possibly be used as an effective biocontrol weapon in the seafood manufacturing industry.

Aroplectrus dimerus (Hymenoptera: Eulophidae), Ectoparasitoid of the Nettle Caterpillar, Oxyplax pallivitta (Lepidoptera: Limacodidae): Evaluation in the Hawaiian Islands.

The stinging nettle caterpillar, Oxyplax (syn. Darna) pallivitta (Lepidoptera: Limacodidae), is a serious invasive pest of agricultural products and a health hazard on the Hawaiian Islands first discovered in 2001. Nursery workers and homeowners have been stung by the caterpillars while handling their plants, especially rhapis palms (Rhapis sp.). Throughout its invaded range, it causes widespread damage, including the many cultivated and native palm species that have grown in Hawaii. Larvae contain urticating hairs that secrete a toxin, causing painful skin swelling and irritation on contact. Horticulture and nursery products impacted by the limacodid pest are estimated at $84.3 million (2018 value). Suppression efforts with pesticides and lure traps were ineffective, and the moth population continued to spread to major Hawaiian Islands (Hawaii, Kauai, Maui, Oahu). The introduction of specific biological control agents from the native region was thought to be the long-term solution for this invasive pest. Initial exploration in Indonesia and Thailand resulted in the introduction of a pupal ectoparasitoid, Nesolynx sp. (Hymenoptera: Eulophidae: Tetrastichinae), that was not specific. The oriental wasp, Aroplectrus dimerus Lin (Hymenoptera: Eulophidae: Eulophinae), idiobiont gregarious ectoparasitoid of the stinging nettle caterpillar, was introduced from Taiwan in 2004 for host specificity studies and biocontrol in Hawaii. Host range testing showed the parasitoid attacked only limacodid species, and it was approved for field release in 2010. The parasitoid identity, host specificity under containment facility conditions, reproductive performance, and colonization on the major infested sites were assessed. A total of 13,379 parasitoids were colonized on 162 release sites on four Hawaiian Islands. Evaluations were conducted using field surveys of larvae, pupal counts, and male lure traps. Field parasitism was thoroughly investigated on Oahu Island, averaging 18.9 ± 5.6% of 3923 collected larvae during 2010-2023. The numbers of male moths caught/trap/month were significantly reduced on Oahu Island (p < 0.05). Recently, the hyperparasitoid, Pediobius imbreus Walker (Hymenoptera: Eulophidae: Entedoninae), was detected, reducing the efficiency of A. dimerus in the field. The mean hyperparasitism of A. dimerus pupae was 27.3 ± 7.6% on Oahu Island. There was no detailed biological assessment for A. dimerus or its field evaluation available in scientific literature. Results were discussed regarding the potential use of A. dimerus in biocontrol elsewhere if the stinging nettle caterpillar was invaded in the future.

Investigating the host finding behaviour of the weevil Phytobius vestitus for the biological control of the invasive aquatic weed Myriophyllum aquaticum

Biological control programmes involve pre-release studies that include host range tests to predict likely negative effects on native species. Behavioural responses of potential biological control agents to olfactory signals emitted by target and non-target species provide information on the host-finding and selection behaviour for safer agent selection. The weevil Phytobius vestitus (Coleoptera: Curculionidae) was identified as a potential biological control agent for the sustainable management of the aquatic weed Myriophyllum aquaticum (Haloragaceae). Using olfactometer-based bioassays, we analysed the host-finding and host-selection behaviour of P. vestitus across a variety of plant species closely related to M. aquaticum using a glass olfactometer. We further analysed the volatile organic compounds emitted by the target and non-target plant species to explain the weevil's observed preferences. We demonstrated that P. vestitus was able to recognise and discriminate between eight closely related Haloragaceae plant species and was significantly more attracted to the olfactory cues emitted by the target plant, M. aquaticum, compared to the other plant species. This study provides a first important indication of the specificity of P. vestitus and shows the role of plant chemistry in host selection and the possibility of refining host range assessment for potential biological control agents. However, exhaustive risk assessment for possible release into targeted new environments still requires further investigations.

Phylogeography, origin and population structure of the self-fertile emerging plant pathogen Phytophthora pseudosyringae.

Phytophthora pseudosyringae is a self-fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping-by-sequencing. Coalescent-based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide.

Isolation and characterization of lytic bacteriophages from various sources in Addis Ababa against antimicrobial-resistant diarrheagenic Escherichia coli strains and evaluation of their therapeutic potential

BackgroundEscherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions.MethodsA total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR).ResultsIn total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels.ConclusionsThe promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections.

Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi.

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

Pectobacterium carotovorum Subsp. brasiliense Causing Soft Rot in Eggplant in Xinjiang, China.

An outbreak of stem rot in eggplants was observed in Heshuo County, Xinjiang, during winter 2021-2022 in about 12-35% of the eggplants in the region (about 40 hm2). The infected tissues yielded a total of four bacterial strains, which were subsequently subjected to physiological and biochemical assays as well as molecular identification. Based on these analyses, the pathogen was identified as Pectobacterium carotovorum subsp. brasiliense. The pathogenicity was confirmed through the fulfillment of Koch's postulates. The host range test confirmed the broad spectrum of species susceptible to infection by the strains. This study represents the first case of infection caused by P. carotovorum subsp. brasiliense resulting in stem rot in eggplant.

Isolation and characterization of multidrug-resistant Salmonella-specific bacteriophages and their antibacterial efficiency in chicken breast

The use of phages as biocontrol agents against antibiotic-resistant strains of Salmonella spp. is gaining attention. This study aimed to isolate lytic bacteriophages specific for multidrug-resistant Salmonella enterica serovars Typhimurium; it also evaluated the bactericidal effect of isolated phages (STP-1, STP-2, STP-3, and STP-4) from sewage sample against S. Typhimurium as host strains. Moreover, a current study evaluated the efficacy of a bacteriophage cocktail against S. Typhimurium cocktail in chicken breast meat. The 4 phages were classified under the Caudoviricetes class by morphology characterization. On host range testing, they exhibited lytic activities against S. Typhimurium, S. Enteritidis, and S. Thompson. In the stability test, the phages exhibited resistance to heat (above 70°C for 1 h) and pH (strongly alkaline for 24 h). Additionally, the phages had comparable adsorption rates (approximately 80% adsorption in under 5 min). Additionally, the latent periods ranged from 30 to 50 min, with respective burst sizes of 31, 218, 197, and 218 PFU/CFU. In vitro, bacterial challenge demonstrated that at a multiplicity of infection (MOI) of 10, each phage consistently inhibited S. Typhimurium growth at 37°C for 24 h. In the food test, the phage cocktail (MOI=1,000) reduced S. Typhimurium in artificially contaminated chicken breast meat stored at 4°C by 0.9 and 1.2 log CFU/g after 1 and 7 d, respectively. The results point toward a promising avenue for addressing the challenge of multidrug-resistant S. Typhimurium in the food industry through the use of recently discovered phages. Notably, the exploration of phage cocktails holds significant potential for combating S. Typhimurium in chicken breast products in the times ahead.

Paenibacillus larvae and their phages; a community science approach to discovery and initial testing of prophylactic phage cocktails against American Foulbrood in New Zealand.

Background: American foulbrood (AFB) is a devastating disease of the European honey bee (Apis mellifera) and is found throughout the world. AFB is caused by the bacterium Paenibacillus larvae (P. larvae). Treatment with antibiotics is strictly forbidden in many regions, including New Zealand. Safe and natural prophylactic solutions to protect honey bees from AFB are needed. Bacteriophages are a well-studied alternative to antibiotics and have been shown to be effective against P. larvae in other countries. Methods: We employed a community science approach to obtaining samples from around New Zealand to discover novel bacteriophages. Standard isolation approaches were employed for both bacteria and bacteriophages. Host range testing was performed by agar overlay spot tests, and cocktail formulation and in vitro testing were performed in 96-well plate assays, followed by sub-sampling and CFU visualization on agar plates. Results: Herein, we describe the discovery and isolation of eight P. larvae bacterial isolates and 26 P. larvae bacteriophages that are novel and native to New Zealand. The phage genomes were sequenced and annotated, and their genomes were compared to extant sequenced P. larvae phage genomes. We test the host ranges of the bacteriophages and formulate cocktails to undertake in vitro testing on a set of representative bacterial strains. These results form the basis of a promising solution for protecting honey bees in New Zealand from AFB.

Isolation of Clavibacter michiganensis subsp. michiganenesis-specific bacteriophages from tomato fields in Turkey and their biocontrol potential

BackgroundClavibacter michiganensis subsp. michiganensis (Cmm) is causing wilt and canker and significant economic losses in tomato production. Current control methods with conventional chemicals or antibiotics lost their effectiveness due to the development of bacterial resistance to this agent. Bacteriophages, viruses of bacteria, have been attracting increasing attention as an environmentally friendly means of controlling bacterial diseases. In this study, Cmm and Cmm-specific lytic bacteriophages were isolated from rhizosphere soil and phyllospheres of diseased tomato plants, and the biocontrol potential of bacteriophages was investigated.ResultsEight isolates that showed typical symptoms of Cmm infection in biochemical and pathogenicity tests contained the virulence gene pat-1 in the genome. Twenty-two native bacteriophages were isolated from 90 samples. As a result of the host range tests performed on the local Cmm isolates obtained in the study, temperature and pH tolerance tests were performed with ten phages with a wide host range. Phage24, Phage33 and Phage41, which have higher temperature and pH tolerance than other phages, were used to kill curve assay. Although the combined applications of phages were performed to increase efficacy, it was found that the application of Phage33 alone was more effective.ConclusionPhage33 may prove to be a suitable agent to be used as part of the microbial control strategy of Cmm. In further studies, Phage33 should be formulated to protect it from adverse environmental conditions and tested under field conditions to confirm the results obtained in this study.

Strategies for the Biological Control of Parthenium hysterophorus L. in Eastern Pakistan

Abstract The arrival of the invasive agricultural weed parthenium, Parthenium hysterophorus , in Pakistan was first noted in the Gujrat district of the Punjab Province in 1980. The plant has now spread further in the Punjab Province as well as into Khyber Pakhtunkhwa (KP), the Islamabad Capital Territory (ICT) and Azad Jammu and Kashmir. The heartlands of parthenium infestation are in the central and northern districts of Punjab, the ICT and KP, and the weed is moving into the southern parts of Punjab and north-eastern KP. Given parthenium’s highly invasive nature, it is likely that it has already spread into the southern parts of Punjab, especially as the area is home to extensive irrigated farming, and it is possible that the plant has even reached Sind and Baluchistan provinces in the south and south-west of Pakistan. To date, two biological control agents for parthenium have reached Pakistan without being introduced deliberately, most likely arriving from India or Nepal: Zygogramma bicolorata – the Mexican or parthenium leaf beetle – and Puccinia abrupta partheniicola , or winter rust. This case study assesses the effectiveness of these existing biological control agents in Pakistan, the potential of redistribution projects to increase their impact and whether artificially inducing Z. bicolorata to emerge from diapause early could further stem parthenium growth in spring. The second part of the case describes the importation of the stem-boring weevil Listronotus setosipennis into a quarantine facility for host range testing. The plan is to boost Pakistan’s biological control programme by releasing the weevil in areas where parthenium occurs. Finally, the case also recommends additional biological control agents and strategies for the future. Adapted from: The biological control of Parthenium hysterophorus L. in Pakistan: Status quo and future prospects'. Management of Biological Invasions (2021), Volume 12, Issue 3: pages 509-526. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Information © CAB International 2023

Evaluation of Bipolaris yamadae as a bioherbicidal agent against grass weeds in arable crops.

Weeds are among the most damaging pests of agriculture, causing ≈10% worldwide reduction in crop productivity each year. Over-reliance on synthetic chemical herbicides has caused weeds around the world to evolve resistance. Bioherbicides may be an alternative. However, among their many constraints including strict environmental requirements, complicated mass-production and high product costs, limited pathogenicity and a narrow spectrum of activity are frequently encountered and are major barriers to commercialization. We isolated a pathogenic fungus, HXDC-1-2, from diseased leaves of a gramineous weed, stiltgrass [Microstegium vimineum (Trin.) A. Camus], from the edge of farmland in Guizhou province, China. HXDC-1-2 was identified as the fungal species Bipolaris yamadae based on the morphological characteristics and ITS-GPDH-EF1α multiple primer analysis. Its potential as a bioherbicide was evaluated by determining its weed control efficacy and crop safety. The ED50 and ED90 values of HXDC-1-2 on Echinochloa crus-galli were 3.22 × 103 and 1.32 × 105 conidia mL-1 , respectively. Host range tests revealed that 20 gramineous weeds including Setaria viridis, Leptochloa chinensis, Eleusine indica, Pseudosorghum zollingeri, Leptochloa panicea, Bromus catharticus, E. crus-galli plants, were extremely susceptible whereas 77 crop species from 27 plant families including rice, wheat, barley, corn, soybean and cotton (excluding cowpea and sorghum) were unaffected. Bipolaris yamadae strain HXDC-1-2 has great potential to be developed as a commercial broad-spectrum bioherbicidal agent for controlling grass weeds in arable crops. © 2023 Society of Chemical Industry.

Genetic structure and host associations of the noctuid moth Callopistria exotica, a potential biological control agent for Lygodium microphyllum

ABSTRACT Lygodium microphyllum is a damaging environmental weed that continues to spread rapidly across Florida. The natural enemies released so far have had some impact on L. microphyllum, but this weed continues to grow densely and spread, so new biological control agents are required. Native range surveys of L. microphyllum identified a noctuid moth, Callopistria exotica, commonly feeding on this fern. We collected C. exotica feeding on Lygodium in Australia, China, India, Indonesia, Japan, Malaysia, Singapore, and Thailand, and sequenced three genes (COI, 18S, 28S) to assess genetic structure across its geographic distribution. We identified a unique C. exotica genetic lineage in Japan, feeding on Lygodium japonicum. This lineage may represent a distinct species, and could potentially be well-suited to control L. japonicum, which has also developed into a damaging weed across Florida. Furthermore, we found genetic structuring within the moth samples collected from L. microphyllum, with three distinct mitochondrial lineages identified. We recommend treating these distinct C. exotica lineages independently from one another for the purposes of host range testing, and suggest further tests to clarify their species status relative to one another. Our study illustrates how the genetic screening of native range survey data can provide important preliminary information that helps develop hypotheses to direct additional surveys, agent selection, and host range testing.

Isolation, identification, and host range of Aspergillus welwitschiae causing postharvest rot on Chinese cabbage in China

A new kind of postharvest rot on Chinese cabbage (Brassica rapa L. ssp. pekinensis) caused by a fungus was observed in vegetable cellars in Harbin, China in 2019 and 2020, causing considerable economic losses. A water-soaked spot appeared at the base of the midrib of leaves, extended into the upper part of the midrib, and ultimately turned into rot. The diseased part was dark brown or black with a few black spores on the surface. Fungal isolates were obtained from the diseased plants and identified as Aspergillus welwitschiae through morphological observation and multigene sequencing analysis of the internal transcribed spacer, β-tubulin and calmodulin genetic regions. Pathogenicity tests were conducted, and the resulting symptoms on Chinese cabbage were similar to those seen in the vegetable cellar. The isolates were also associated with rot and leaf spot on seedlings and affected the seed germination of Chinese cabbage. A host range test showed that the isolates could infect some common vegetables, including carrot, kidney bean, broccoli, radish, Chinese flowering cabbage, root-mustard, cabbage, non-heading Chinese cabbage, pepper, lettuce, oilseed rape, tomato and cucumber. The isolation, identification, and host range of the pathogen can provide a basis for the study of the occurrence, prevention and management of the disease in the future.

Population genetics comparison of Lilioceris cheni (Coleoptera: Chrysomelidae) colonies released onto Dioscorea bulbifera in Southeastern U.S.A.

ABSTRACT Multiple importations of Lilioceris cheni, a defoliating beetle of the invasive air potato plant, were received by the ARS-Invasive Plant Research Laboratory from 2002 to 2012. The last two, in 2011 (China) and 2012 (Nepal), formed the basis of two colonies from which releases were made into six Southeastern U.S. States. Colony populations were examined using the mitochondrial COI sequence. Phylogenetics, evolutionary divergence, a haplotype network, population statistics, and migration models were generated for the two colonies, countries of origin, and the inferred populations (clades). Phylogenetics eliminated the possibility of cryptic speciation and alleviated the need for host-range testing of the 2012 Nepalese samples. Evolutionary divergence showed the Chinese colony was 1.65X as divergent as the Nepalese, but one Nepalese clade was the most distinctly different of all clades. AMOVA showed most (>70%) genetic variation resided within rather than between colonies. In contrast, AMOVAs showed high levels (>61.5%) of genetic variation between underlying clades with proportionally less variation within. The haplotype network showed broad agreement with the phylogeny. Clade C, from China, displayed the largest number of haplotypes and the largest mutation-scaled effective population size in MIGRATE software. The best MIGRATE models indicated that migration and descent followed the order of phylogenetic descent. The results suggest that the genetic diversity being offered to the adventive range by these very divergent colonies (and clades) is large and should support great ecological flexibility.

First Report of Macruropyxis fraxini Causing Rust Disease on Microstegium vimineum in China.

Stiltgrass (Microstegium vimineum (Trin.) A. Camus) is an annual Poaceae weed with a broad native range throughout East Asia. Stiltgrass is an invasive grass that is distributed in more than 15 provinces in China, posing a major threat to native biodiversity and restoration efforts in introduced areas. Stiltgrass often forms dense near-monocultures in forest understories and riparian areas where it disrupts forest succession, nitrogen cycling, and alters native communities (Stricker et al. 2016). In August 2018, M. vimineum with rust disease were observed near the roadside (26.759482 °E, 114.283519 °N) in Jinggangshan City, Jiangxi Province, China. Diseased plants were observed at a 2 × 10 m shady location with lesions on leaves and stems, disease incidence was over 90% (n=100). Sixty disease samples were collected to confirm the pathogen. Early symptoms on the upper leaf surfaces consisted of rust pustules, which were circular, subcircular to irregular, orange to dark-orange, crust-like, and granular. At later stages, lesions coalesced, spreading all over the plant, causing severe defoliation. Uredinia were predominantly formed on the upper surface of leaves and young stems but rarely also found on the abaxial leaf surface, exposed, yellow to yellow-orange, and 0.2-0.5 mm in diameter, occasionally reaching 0.9-1 mm, surrounded by purple lesions (n=30) (Olympus SZX7). Telia were predominantly formed on the lower surface of leaves, stems, exposed, chestnut-brown to dark-brown. Urediospores were nearly spherical, oval or obovate, light yellow, 18-23 μm × 20-26 μm, cell wall is about 2-2.5 μm (n=200) (ZEISS AXIO Imager. M2). Teliospores were ellipsoid, 37-55 μm × 25-36 μm, 2-celled, inner wall brown, 4-5.5 µm thick, outer wall hyaline, smooth, germ pores 2-4 per cell; pedicels were hyaline, composed of cell walls with loss of cytoplasm, 4.5-6.5 µm wide, and up to 160 µm long (n=200) (ZEISS AXIO Imager. M2). Pycniospores, aeciospores and basidiospores were not observed in this study. The telial morphology features were consistent with those reported of M. fraxini, but uredinial stages were not observed in these studies (Azbukina 1974; Jung et al. 2020). Genomic DNA was extracted from a representative specimen (JGS-1) and was characterized by PCR amplification and sequencing of 28S rDNA using the primer pair NS1 and NS4 (Aime 2006). The 1094-bp sequence (Genbank: ON739170) shared 99.18% nucleotide identity with M. fraxini (Genbank: KP858144). The internal transcribed spacer (ITS) region was sequenced by rust fungal primer pairs ITS4rust and ITS5u (Pfunder et al. 2001). The 564-bp sequence (Genbank: ON739169) shared 99.12% nucleotide identity with M. fraxini (Genbank: KP858145), which was consistent with the morphological features observed. To complete Koch's postulates, plants were inoculated by brushing a urediniospore suspension (1 ×105 spores/ml) onto the leaves, placed in a plant growth chamber (25℃, 8 h/d of dark, 30℃, 16 h/d of light, 8000 lux of light intensity, RH ≥ 90%). Urediniospores were formed on the leaf surface 7 to 10 days after inoculation, and all infected plants showed symptoms similar to those observed in the field, along with spores, whereas the control plants remained symptomless. Host range tests showed that rice, wheat, barley, sorghum, maize, cotton, peanut and rape were resistant to M. fraxini but soybean and peas were susceptible. More research is needed to determine whether this pathogen can be a biocontrol agent for stiltgrass, such as exploring the potential impact of this rust pathogen, expanding host range tests, and finding its alternate hosts. To the best of our knowledge, this is the first report of rust disease on stiltgrass caused by M. fraxini in China.

Pathogenicity and host range of Pseudocercospora fumosa, a potential biological control agent for moth plant (Araujia hortorum) in New Zealand

ABSTRACT The South American vine Araujia hortorum (Apocynaceae) was introduced as an ornamental species in several countries and has since become an invasive weed in many of them. In New Zealand it has been targeted for classical biological control, and this motivated field surveys for fungal pathogens in its native range in Argentina. A disease was frequently encountered in the field causing extensive damage to foliage, as well as stems and fruits. The fungus found in association with the symptoms was identified as Pseudocercospora fumosa. A detailed description of the fungus and the disease is provided. Pathogenicity tests were conducted, and Koch’s postulates fulfilled. Host range tests were conducted on species within the Apocynaceae. Four of these, belonging to the same subtribe as A. hortorum, were found to be susceptible to the disease, while others less related ones were not. Pseudocercospora fumosa was observed to be able to cause severe disease on its host and to be highly specific. It is therefore proposed as a potential biological control agent against A. hortorum in New Zealand.