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Francesco Paoli (male) obtained a PhD in Evolutionary Biology at the University of Siena in 2010. During his PhD, he learned techniques of transmission and scanning electron microscopy, corrosion casting, microCT and 3D reconstruction. Since May 2012, he has been working at the Council for Agricultural Research (CRA ABP), nowadays CREA DC, where he holds a permanent position as technologist since 2019. In this period he has dealt with the biological/low-impact control of Popillia japonica and other alien pests and investigated biological traits of quarantine insects, such as Rhynchophorus ferrugineus, Matsucoccus feytaudi¸ and Planococcus ficus. He is involved in the national project OliDiXIIt (Olive-growing and protection from Xylella fastidiosa and vector insects in Italy, 2018-2021). In July 2018, he obtained the national scientific qualification (ASN) as second level professor in Plant Pathology and Entomology.

Leonardo Marianelli (male) graduated in Forestry and Environmental Sciences at the University of Florence in 2001. In 2004, he obtained a PhD in "Agricultural Microbial Biotechnology". During the PhD he learned concepts about Forest pathology, cell biology techniques, diagnosis method for major diseases. Since 2005 he has been working at the Council for Agricultural Research (CRA ABP), which is nowadays CREA DC. He was involved in various national projects (RISALE, PINITALY, BIOCONTROL, LABCO) as inspector of the main entomological and phytopathological pests in forest areas. From October 2012 to February 2014, he acquired the qualification for the profession of Phytosanitary Inspector at the Tuscany Region Plant Protection Organization. In this period he improved knowledge of relevant EU legislation and the relevant International Standards for Phytosanitary Measures (ISPMs) and European Plant Protection Organization (EPPO) standards. Since March 2014, he holds a position as a permanent researcher at CREA DC in Florence, where he has been involved in many projects about alien pest detection and control. In 2016, he became Member of the National Technical Table for Popillia japonica phytosanitary emergency (D.G.R. n. 22-2865 of 02/01/2016 and D.D. n. 73 of 12/02/2016). From January 2016 to 2018, he was coordinator of the COBIPO project “biological control of Popillia japonica” by means of entomopathogenic nematodes and fungi, and by means of microbials and low-impact chemicals.

Background and Aims

Popillia japonica is a scarab beetle native to Japan that in the last century has spread into the US, Canada, The Azores, Italy and, transiently, Switzerland. Due to its potential harmfulness to agricultural crops, as well as landscape plants, this insect is currently included within the EU priority pest list. Popillia japonica is highly polyphagous, being able to feed on more than 300 plant species, and highly mobile, capable of flying for several kilometers every day. Moreover, it can spread through the movement of goods and people. In Italy, after its first discovery in 2014, this pest has spread into an area of about 6,000 square kilometers, thus confirming its invasiveness. This insect damages plants both at the larval and adult stage: larvae feed on the roots of turf and pasture grasses and row crops (e.g. maize), whereas adults feed on many woody and herbaceous plants by eating leaves (e.g. hazelnuts, grapevine), flowers and fruits (e.g. soft and stone fruits). In the US about $460 million are expended every year to cover the cost of the management of the Japanese beetle and the replacement of damaged plants. Analysis of climatic suitability shows that most of the European countries are suitable for Japanese beetle establishment. So, it is likely that without an effective way to slow its spread, P. japonica could soon become a problem in several EU countries. Control methods for this pest have often included the use of chemicals in the US. However, the growing concern about the environmental pollution caused by pesticides is promoting research on the use of biological control agents, such as parasitoid insects, entomopathogenic bacteria, fungi and nematodes, and on other eco-friendly alternative control systems. Recently, a strategy has been developed based on the technology of Long-Lasting Insecticide-treated Nets (LLINs). A new attract-and-kill device - consisting of a net impregnated with a pyrethroid and standard pheromone and kairomone dual-lures - has been tested and extensively employed in Italy to limit the spread of this pest. In the present study we show the results of lab to field experimentation in which we evaluated the effectiveness of two different LLINs by considering the number of dead and paralyzed beetles as a function of exposure time on the net. We also evaluated the duration of effectiveness of the attract-and-kill devices when exposed outside for the entire flight period, which is roughly from June to September

Methods

Adult beetles were collected from infested areas in the municipality of Cameri (Piedmont, North East Italy) and experiments were carried out at the local entomological laboratory of the Ticino Valley Park. Laboratory experiments were conducted by allowing beetles to walk on two different LLINs: Storanet (BASF, Germany) and ZeroFly (Vestergaard, Switzerland). Storanet contained α-cypermethrin at a dose of 1.57mg AI/g fiber, commercialized in Austria to control forest pests (e.g., Ips typographus L.). The ZeroFly contained deltamethrin at a dose of 3.85mg AI/g fiber. The lethal effects of both LLINs were tested in the lab by exposing adult beetles to the nets for 5 sec, 15 sec, 30 sec, 1 min, 5 min, 15 min, or 30 min (N = 100 insects per net/timing combination). The insects were then held in ventilated plastic boxes with food for 13 days, their health condition daily registered.

To verify the killing ability of the attract-and-kill device through the season, toxicity tests were conducted in the lab using field exposed LLINs. The attract-and-kill devices were applied in four fields with similar characteristics, roughly 500 m from each other, within the infested area in the municipality of Cameri. In each field, four attract and kill devices plus one LLIN without pheromone were positioned. From June till the end of September, every month one attract-and-kill device from each of the fields was removed and the net used for toxicity tests. In these tests 20 adults per treatment were allowed to walk on the removed nets for 90 sec, which is the observed mean value of insect permanence on the attract-and-kill devices in the field. Treated insects were then reared as described above and their health condition registered.

Results / Outcomes

Results from lab trials showed that both LLINs were effective in killing the beetles; however, some differences emerged when diverse exposure times were compared: ZeroFly always presented 100% mortality in tests from 30-min to 5-sec exposure, whereas Storanet caused 100% mortality only with 30-min exposure, decreasing to 99–89% mortality for 15-min to 5-sec exposure.

As for field experiments, we observed that after the first month exposure outside the attract-and-kill devices remain effective, although with performance reduction of about 30%. In the following months, the effectiveness appears to progressively decline. However, increasing natural mortality of Japanese beetles late in the growing season may compensate for decreased effectiveness of the LLIN in August and September. More dedicated experiments are needed to clarify this topic.

Conclusions

In conclusion, our data suggest the possible use of attract-and-kill devices based on LLIN technology as part of Integrated Pest Management programmes to control the Japanese beetle, and give a practical indication on the duration of the effectiveness of these devices. The use of attract-and-kill devices provides useful advantages in the management of P. japonica outbreaks: the system is effective throughout the season and compared to mass catching with traps it is easier to manage because it does not require the continuous emptying of collecting containers.

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Pepori Alessia Lucia

Graduated in Management of Forest Systems, she obtained the PhD in Agricultural Microbial Biotechnology (University of Florence), continuing her study of the role and interaction of Gen. Geosmithia in relation to the elm-grafiosis elm pathosystem. Presently, her research focuses on the study of invasive forest pathogens. The study of genetic variability of forest pathogen populations and the study of induced systemic resistance caused by root and foliage pathogens, will improve the knowledge about the host physiological responses, the effects of pathogenic fungi and the genetic changes in native organisms phylogenetically related to invasive species.

Main research topics:
• Invasive forest pathogens: identification and determinants of invasion. Interaction between alien and native pathogen.
• Real‐time PCR assay optimization to detect and quantify DNA from different pathogens in plant and soil and insects.
• Transcriptome analysis by Real-time PCR assay to study the interaction host plant/pathogen.
• Fungus‐fungus interactions (Vegetative Compatibility, association, parasitism, etc.)
• Host‐pathogen‐environment interaction.
• Plants and pathogens population genetics.

Alberto Santini

Interests and present fields of work

• Invasive species. Biogeographic patterns and determinants of invasion by alien forest pathogens in Europe; early detection of invasive pathogens; impact of climate change on pathogens invasion processes
• Host-pathogen-environment interaction. Genetic variability of disease resistance, Mechanisms of resistance to pathogens, mechanisms of disease escape and disease avoidance.
• Genetics of pathogens populations.
• Breeding for resistance to pathogens. Selection of trees resistant to pathogens by controlled crossing and artificial inoculations. Adaptative trials.
• Conservation of genetic resources of endangered species, mainly focus on elm. Studies on adaptative characters connected with resistance to pathogens. Genetic studies on trees marginal endangered populations.

Background and Aims

In Europe as in North America elms are devastated by Dutch elm disease (DED), caused by the alien ascomycetes Ophiostoma ulmi and O. novo-ulmi (ONU). Pathogen dispersal and transmission are ensured by local species of bark beetles (Coleoptera: Curculionidae, Scolytinae), which established a novel association with the alien fungi. Elm bark beetles also transport fungi of the genus Geosmithia, that are commonly found in scolytids’ galleries colonized by ONU. Widespread horizontal gene transfer between ONU and Geosmithia was recently observed.

In order to define the relation between these two fungi in the DED pathosystem, ONU and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. We also study the presence of the two pathogens on the body of vector insects, and set up a duplex real time PCR to detect in same sample the quantity of fungi to better undestand the biological cycle of Geosmithia and the natural interaction with Ophiostoma.

Methods

O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. We also study the presence of the two pathogens on the body of vector insects, and set up a duplex real time PCR to detect in same sample the quantity of fungi to better undestand the biological cycle of Geosmithia and the natural interaction with Ophiostoma.

Results / Outcomes

Growth and sporulation of ONU resulted significantly higher in the presence of Geosmithia than in its absence. Signs of mycoparasitism by Geosmithia on ONU hyphae, such as formation of coilings, appressoria-like branches, pseudopod-like structures or short hooks, were commonly observed at microscopy. Finally, the addition of spores of Geosmithia species to suspensions of ONU spores used for artificial inoculations significantly reduced DED symptoms. We observed a close relationship in the presence of the two fungi on the body of bark beetles, in relation to different levels of infection of the diseases analyzed in the natural environment.

Conclusions

A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on ONU. Studies to determine the feasibility of a classical biological control action are ongoing by observing life cycles of the different organisms implied in the association and how they are interconnected in vivo, as well as their in vitro interactions.

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Mwafaq Ibdah has completed his PhD at the age of 32 years from Martin Luther University, Hale/Salle, Germany and postdoctoral studies from Michigan State Universtity and Wahsingion State University. He is tenured researcher at Newe Ya’ar Research Center, The Agriculture Research Organization (ARO), in Isarel . He has published more than 20 papers in reputed journals and has been serving as an editorial board member of repute.

Background and Aims

Pear Psylla is the most important pest of pear in all pear-growing regions, in Asian, European, and the USA. Pear psylla damages pears in several ways: High-density populations of these insects can cause premature leaf and fruit drop, diminish plant growth, and reduce fruit size. In addition, their honeydew promotes sooty mold on leaves and russeting on fruit. Pear psyllas are also considered vectors of pear pathogens such as Candidatus Phytoplasma pyri causing pear decline that can lead to loss of crop and tree vigor, and sometimes loss of trees. Psylla control is a major obstacle to efficient Integrated Pest Management.

Methods

GC-MS volatile metabolic profiling identified several volatile compounds common in these accessions but lacking, or much less common, in a sensitive accession, the commercial Spadona variety. Among these volatiles were styrene and its derivatives. When the resistant accessions were used as inter-stock, the volatile compounds appear in commercial Spadona scion leaves and it showed reduced susceptibility to pear psylla. Laboratory experiments and applications of some of these volatile compounds were very effective against psylla eggs, nymphs and adults.

Results / Outcomes

The genes and enzymes involved in the specific reactions that lead to the biosynthesis of styrene in plant are unknown. We have identified a phenolic acid decarboxylase that catalyzes the formation of p-hydroxystyrene, which occurs as a styrene analog in resistant pear genotypes. The His-tagged and affinity chromatography purified E. coli-expressed pear PyPAD1 protein could decarboxylate p-coumaric acid, and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene. In addition, PyPAD1 had the highest activity towards p-coumaric acid.

Expression analysis of the PyPAD gene revealed that its expressed as expected, i.e. high when styrene levels, and psylla resistance were high.

Conclusions

In this study, levels of styrene and p-hydroxystyrene were found to be elevated upon grafting psylla-sensitive cultivars on resistant rootstocks. In addition, the biological activity of styrene and p-hydroxystyrene was found to be highly effective against juvenile forms of psylla and dramatically reduced their numbers. A new PAD gene/protein has been identified from pear leaves. The His-tagged and affinity chromatography purified E. coli-expressed pear PyPAD1 protein could decarboxylate p-coumaric acid , and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene . In addition, PyPAD1 had the highest activity towards p-coumaric acid . To the best of our knowledge, this is the first report of the characterization of a phenolic acid decarboxylase involved in p-hydroxystyrene formation in pear. This work also provides insight into the metabolic grid involved in the biosynthesis of styrene analogs.

The novel PyPAD1 encoding gene here could be used for developing molecular markers associated with resistance to pear psylla based on styrene analogs and other metabolic factors. These results can be useful for world-wide breeding of pear cultivars that require significantly less use of insecticides harmful to human health and to the environment.

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Dr Nawaporn Onkokesung

I am a plant molecular biologist with a specific interest in plant interactions with environmental conditions and adaptive response to environmental stress. Since 2016, I have been working as research associate in Professor Robert Edwards’ group to decipher molecular mechanisms of herbicide resistance in grass weeds. I am one of the five listed inventors of the ‘Non-Target site Resistance’ patent application.

Follow my researches and outreach activities @NOnkokesung

Professor Neil Boonham

Professor Boonham is a plant pathologist and molecular biologist with 25 years of experience in the development and deployment of diagnostic technologies to help decision making in the plant health and agricultural sectors. Neil led the development of the herbicide TSR diagnostic tests based on LAMP amplification. He has spent significant amounts of time demonstrating both the TSR and NTSR tests to farmers and agronomists in the UK, USA and Canada to evaluate the perceived benefits and drawbacks to the approach and assess the needs of the market place.

Follow his researches and outreach activities @neilboonham

Background and Aims

Herbicide application is a common and cost- effective approach to control agricultural weeds. However, the overuse of herbicides is a major factor leading to the evolution of herbicide resistance in various weeds species. Herbicide resistant black-grass populations are widespread in the UK and western Europe including France, Germany and the Netherlands. In the UK, herbicide resistant black-grass incurs an economic cost of ~£0.5bn/year and is associated with 1-million-ton/year yield loss in wheat production¹.

Target site (TSR) and non-target site (NTSR) resistances are the two known mechanisms conferring resistance to herbicides in various weed species including black-grass (Alopecurus myosuroides), rye-grass (Lolium rigidum), and wild oat (Avena fatua). TSR confers resistance to specific herbicide chemistry due to the mutations of the target proteins that alter herbicide-targeted protein structures. Hence, herbicides are unable to bind to the target proteins. The herbicide resistances occurring without mutations at the target proteins are collectively referred to as NTSR. At present, growers send samples of seed to laboratories to identify herbicide resistance by bioassay using petri-dish or spray assays. These methods can take up to 3 months. Even though this bioassay provides accurate information of herbicide resistance in weeds, this assay cannot pinpoint the resistant mechanisms.

Acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibitors are two common herbicide modes-of-action used to control grass weeds including black-grass in cereal crops. To date, the mutation at proline 197 (Pro 197) and tryptophan 574 (Trp 574) in ALS and the mutation at isoleucine 1781 (Ile 1781) in ACCase are the most common mutations conferring resistance to ALS inhibitors (i.e. sulphonyl urea) or ACCase inhibitor (i.e. aryloxyphenoxypropionates and cyclohexanedione) herbicides, respectively. To detect these mutations, the sequencing of DNA samples needs to be done in a well-equipped laboratory.

In this study, we developed an easy-to-use diagnostic assay that can be done on farms to detect these common mutations in ALS and ACCase genes in grass weeds. The mutations are detected using Loop-mediated Isothermal Amplification (LAMPs) based assay that can provide the results within 45 minutes. We use black-grass as a model system to verify the LAMP assay since the correlations between specific mutations and the resistant to specific herbicide chemistry have been well-documented in this species. Furthermore, the complete DNA sequences of ALS and ACCase genes are publicly available. Early detection of the herbicide resistance status of a black-grass population prior to spraying will provides growers with information to tailor a site-specific weed management program to improve control, reduce cost and improve crop productivity.

Methods

The specific primers to detect ALS or ACCase mutations were designed using the DNA sequence of ALS (accession number AJ437300) and ACCase (accession number AJ966411) from black-grass.

To verify the LAMP assay, black-grass populations that had been previously confirmed to have mutations at Pro 197 or Trp 574 in ALS or Ile 1781 in ACCase enzyme ² were used. Leaf tissue of the 3-leaf growth stage (GS13) black-grass was homogenized in 750 µL of alkaline polyethylene glycol (pH 13.5) by shaking for 45 seconds. Leaf extract (5 µL) was added to reaction mix (20 µL) containing LAMP reaction buffer and specific primers to detect a specific point mutation in the ALS or ACCase gene (OptiGene, UK). The reactions were run on a Genie III instrument (OptiGene, UK) for 45 minutes. The mutations were identified based on the shifting of melting temperature of the amplification product compare to the melting temperature of the ALS or ACCase product amplified from an authentic wild type sample without mutation.

The field-collected black-grass populations that have been previously tested for herbicide resistance by spray or petri-dish assay were next tested by the LAMP assay. Leaf tissues of field-collected population black-grass at GS13 were used for analysis. The mutations in ALS or ACCase detected by the LAMP assay and the resistance to specific herbicide chemistry of these black-grass populations were analysed to verify the accuracy of the LAMP assay on detecting specific mutations conferring resistance to specific ALS or ACCase inhibiting herbicides.

Results / Outcomes

The LAMP assay was able to detect the specific mutations at Pro 197 or Trp 574 in ALS or at Ile 1781 in ACCase genes from the leaf extract of black-grass populations that had been previously confirmed to carry the mutations. Furthermore, the LAMP assay was able to detect the mutation at Pro 197 of the ALS gene and at Ile 1781 of the ACCase gene in the filed-collected black-grass populations. The mutations in ALS or ACCase genes detected by the LAMP assays correlated with the resistance to sulphonyl urea (mesosulfuron+iodosulfuron) and aryloxyphenoxypropionates (clodinafop) herbicide observed in the field-collected black-grass populations.

Conclusions

The LAMP assays were capable of detecting the specific mutations of ALS or ACCase enzymes conferring resistance to specific herbicide chemistry in black-grass populations. An easy-to-use protocol, rapid detection and an accurate identification of the specific point mutations make LAMP assays a promising tool for in-field testing to detect specific TSR mutations in grass weeds. Furthermore, the flexibility of LAMP technology makes this assay easy to adapt to detect the mutations in other genes that confer herbicide resistance in black-grass and other weed species.

References

1. Varah, A., Ahodo, K., Coutts, S.R. et al. The costs of human-induced evolution in an agricultural system. Nat Sustain (2019) doi:10.1038/s41893-019-0450-8

2. Marshall, R., Hanley, SJ., Hull, R., & Moss, SR. The presence of two different target-site resistance mechanisms in individual plants of Alopecurus myosuroides Huds., identified using a quick molecular test for the characterisation of six ALS and seven ACCase SNPs. Pest. Mang. Sci. (2012) doi: 10.1001/ps.3429