Field observations on phenological phases (supported also by agronomic and meteorological data) of P. spumarius in olive orchards/other agroecosystems

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1 Field observations on phenological phases (supported also by agronomic and meteorological data) of P. spumarius in olive orchards/other agroecosystems (examples by IPSP-CNR e DMMT Università di Brescia) In order to obtain information on the development and ecology of individuals (and of cohorts) and populations of P. spumarius, as well as information on the abundance of different vector and potential vector species of X. fastidiosa in olive orchards and other agroecosystems in Balearic islands: Macrocosm: field surveys in Balearic Islands in different locations (in order to cover as much as possible the environmental variability encountered by P. spumarius/other potential vectors in these areas) Microcosm: small cages for the study of the prolificacy of single individual couples (a male and a female of P. spumarius) in Balearic islands The protocols applied for the research work at the two different levels are described below. OPEN FIELD (OLIVE ORCHARDS or different agroecosystems /environments ALSO DEFINED AS MACROCOSMS) SAMPLING METHOD Choice of the sampling agroecosystem (e.g. olive orchard, almond orchard, vineyard, or natural environment) Characteristics of the selected fields Size: 1 ha (when larger we consider 1 ha regular surface included in a larger field) Presence of weed cover No application of synthetic insecticides Position and climate Figure or the different macrocosm it is advised when feasible to choose agroecosystems characterized by different climatic conditions. Insect population abundance In order to ensure that spittlebugs are abundant enough to guarantee a reduced error in population parameters estimates and to contain the number of needed sampling units, for the first two surveys it is advised to inspect more candidate locations and to finally select those with more abundant spittlebugs populations Sampling units The primary sampling unit (PSU) is represented by the sampled orchard/agroecosystem

2 The minimal space unit where sampling takes place represents the secondary sampling unit (SSU). We have 3 kind of SSU: SSUp area of herbaceous vegetation for the sampling of nymphs SSUa area of herbaceous vegetation for the sampling of adults SSUo olive tree for the sampling of adults SSUs shrub for the sampling of adults Sampling variables Definition of life stages (single or pooled) E (egg, in egg masses) N1 first nymph instar (E and N1 will be pooled and considered together for practical purposes) N2-N3 (second and third instar nymphs, inside the spittle) N4-N5 (fourth and fifth instar nymphs, inside the spittle and characterized by the presence of wing pads) A (adults, males and females) Estimate of population abundance Population density The counting of the number of nymphs per SSU (area of 0.25 m 2 delimited by a wooden frame, figure 1) in the herbaceous vegetation allows estimating a density. The counting of adults per SSU (4 swept) in the herbaceous vegetation allows estimating the number of adults per swept The counting of adults per SSU (10 swept) on an individual tree allows estimating the number of adults per tree Sampling details Nymphs All the plants within the SSUp are inspected for nymphs, each nymph is assigned to a phenological class and all the nymphs of each class are counted The position of the nymphs on the host-plant is recorded (bottom third, medium third and upper third) Herbaceous host-plants Herbaceous plant community within the SSUp is defined based on the two most dominant species (plant species are identified directly in the field when possible,

3 otherwise plant samples are brought to the laboratory and/or with digital pictures of the plant are taken) Phenology of the herbaceous host-plants 3 phenological classes have been defined for the herbaceous plants at the time of insect sampling: growth phase (pre-flowering), flowering phase, post-flowering phase. 2 phases of growth were defined: presence or absence of new tender shoots 2 phases of water stress were defined: presence or absence of water stress Adults on the herbaceous cover The operator makes 4 paths of about 70 cm, each path being accompanied by a swept. Adults are counted, sexed, identified and immediately released (conservative sampling) Adults on trees (eg.olive trees) 10 swept per tree (SSUo) Adults are counted, sexed, identified and immediately released (conservative sampling) Phenology of olive Phenology of olive trees at the time of insect sampling is determined according to Growth stages of mono-and dicotyledonous plants BBCH Monograph (2001) Adults on other trees 10 swept per tree (SSUo) Adults are counted, sexed, identified and immediately released (conservative sampling) Phenology of trees Phenology of other trees at the time of insect sampling is determined according to Growth stages of mono-and dicotyledonous plants BBCH Monograph (2001) Adults on shrubs (or spontaneous woody plants, lentisk, evergreen and deciduous oaks ) 10 swept per tree (SSUo) Adults are counted and identified and immediately released (conservative sampling) Timing of samplings Every 10 days from themid winter until the beginning of summer in order to describe the stage-structure of the population over time. Every two weeks later in the season, when only adults are present. All the methods described so far refer to the sampling of Philaenus spumarius. For the other xylemsap feeder species (Cercopoidea and Cicadellinae) the same technique is applied except that sampling of adults is not conservative and specimen are brought to the lab under ethanol for species identification, if needed.

4 Meteo data recording Oviposition cages Data logger with probes for air temperature and humidity are hanged at the center of the four PSU for the whole period. Probes are shaded and placed at cm from the ground. Meteo data are also available from the closest meteo station and will be compared with our data or they will replace our data in case the data logger is out of order. MICROCOSM (CAGES FOR INSECT OVIPOSITION) 20 cages have been established in Torino and 20 in Bari. IKEA cages made of fine nylon net, cylindrical (Ø 45 cm, height 50 cm) were chosen (figure 3). Inside the cages there is one pot (Ø 30 cm) with one plant of Polygala myrtifolia, a tuft of alfalfa (Medicago sativa) and some hale to favor oviposition. A male and a female of P. spumarius were introduced in the microcosm at the beginning of September in Torino and at the end of September in Bari. The microcosms were maintained in open-air conditions and periodically inspected to check the survival of the insects. Egg counting is currently ongoing: hale is observed with a magnification lens (2-3 x), the whole plants and the pots are visually inspected for the presence of egg masses. Once an egg mass is identified, individual eggs are counted under a stereomicroscope. Number of egg masses, number of egg per egg mass, location of the egg mass (hale, dry leaves, green leaves, pot) are recorded. After counting of eggs, all egg masses are re-introduced in the cage to allow the development of nymphs.

5 Figure 1. Wooden frame of 1 x 0.25 m used for counting the spittlebug nymphs in the herbaceous vegetation in the macrocosm (photo by IPSP-CNR, Italy) Figure 2. An adult of Philaenus spumarius observed on an olive stem (photo by IPSP-CNR, Italy) Figure 3. Outside and inside view of the oviposition cages (microcosms) (photo by IPSP-CNR, Italy)

6 Examples of field data collection forms (images by IPSP-CNR e DMMT Università di Brescia) Date Site Phenological stage Sample (SSUp) % grass cover grass height Vegetation data dominant plant species_1 and % of cover dominant plant species_2 and % of cover plant species with spittles ID plant Legend plant phenology Water stress Spittle position Juvenile instar Preflowering = PreF; Flowering=F; Postflowering = PostF Yes/No Basal=B; Medium=M; Apical = A 1st instar = N1; 2nd and 3rd = N2-N3; 4th and 5th = N4-N5; Adult = Ad Spittle data spittle position on plant phenology Water stress plant Spittlebug Num. of insects Juvenile instar species per spittle PreF F PostF yes no B M A N1 N2-N3 N4-N5 Ad NOTES Date Hour SSU types: sample on: Site SSUa Grass cover Legend Phenological stage Weather SSUo Olive SSUs Shrubs sample on.. Vegetation data Spittlebugs data grass % grass dominant % cover of dominant % cover of Water Other SSU. Philaenus Neophilaenus Aphrophora Notes height cover plant dominant species_1 plant dominant species_2 stress spittlebugs Tot 1 2 3