First Report of Phyllactinia Chubutiana, Nom. Cons. on Vallesia Glabra (Apocynaceae) in Mexico

Botany

First report of Phyllactinia chubutiana, nom. cons. on Vallesia glabra (Apocynaceae) in Mexico

Journal: Botany

Manuscript ID cjb-2020-0211.R1

Manuscript Type: Note

Date Submitted by the 11-Dec-2020 Author:

Complete List of Authors: Félix-Gastélum, Rubén; Universidad de Occidente Leyva-Madrigal, Karla ; Universidad de Occidente Quiroz-Figueroa, Francisco ; Instituto Politécnico Nacional Rodriguez-Mora, Norma; Instituto Politécnico Nacional Maldonado-Mendoza,Draft Ignacio ; Instituto Politécnico Nacional Espinosa-Matías, Silvia; Universidad Nacional Autónoma de México Mora-Romero, Guadalupe; Universidad de Occidente,

Keyword: Powdery mildew, pearl berry, phylogeny, ITS

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

First report of Phyllactinia chubutiana, nom. cons. on Vallesia glabra (Apocynaceae) in

Mexico

Rubén Félix-Gastélum1*, Karla Yeriana Leyva-Madrigal1*, Francisco Roberto Quiroz-

Figueroa2, Norma Rodriguez-Mora2, Ignacio Eduardo Maldonado-Mendoza2, Silvia

Espinosa-Matías3, Guadalupe Arlene Mora-Romero1.

1 Universidad Autónoma de Occidente, Unidad Regional Los Mochis, 81223, Los Mochis,

Sinaloa, México.

2 Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el

Desarrollo Integral Regional (CIIDIR)- Unidad Sinaloa, 81101 Guasave, Sinaloa, México.

3Universidad Nacional Autónoma de México, Facultad de Ciencias, Coyoacán, México, D.F.

México CP 04510 Draft

*These authors contributed equally to this work.

Guadalupe Arlene Mora-Romero, Unidad de Investigación en Ambiente y Salud-Unidad

Regional Los Mochis, 81223, Los Mochis, Sinaloa, México, +52 6688161050, fax +52

6688161001, [email protected]

Competing interests: The authors declare that there are no competing interests.

1 © The Author(s) or their Institution(s) Botany Page 2 of 24

Abstract A new record of the powdery mildew Phyllactinia chubutiana, nom. cons. (= Oidium insolitum, Ovulariopsis insolita) was documented on pearl berry (Vallesia glabra). No teleomorph was observed. This novel record was identified as a member of Ovulariopsis based on the morphological characteristics of the anamorph (hyphae, conidiophores and conidia), and its hemi-endophytic mycelium in V. glabra correspond to P. chubutiana, nom. cons. A Phyllactinia-specific primer pair was designed for the ITS region of the nuclear rDNA. Phylogenetic analyses confirmed the identity of the powdery mildew specimens found in V. glabra in Sinaloa, Mexico. This is the first ever report of this powdery mildew in

V. glabra, and the second report world-wide on a member of the Apocynaceae. Draft Key words: Powdery mildew, pearl berry, phylogeny, ITS.

2 © The Author(s) or their Institution(s) Page 3 of 24 Botany

Introduction

Powdery mildews adversely affect a range of agricultural and ornamental plants, and

the causal agents are biotrophic, obligate parasites (Bushnell 2002). Powdery mildew (PM)

fungi disseminate via conidia which are favored by high humidity, but not by rain or

immersion in water (Sivapalan 1993a; 1993b). They are members of the family Erysiphaceae

in the order Helotiales, and parasitize around 9,838 angiosperm species (Amano 1986); 93%

of host plants are dicotyledons, while 7% are monocotyledons (Takamatsu 2013).

The mycelium of PM fungi is usually epiphytic, except in the genera Leveillula,

Phyllactinia, Pleochaeta, and in one species of Cystotheca in which the mycelium is hemi-

endophytic. In the latter case, the pathogen produces superficial hyphae that penetrate the leaves through the stomata and produceDraft mycelia inside, in contrast to Leveillula species where mycelia are abundant within the leaf of the host plant (Braun et al. 2002).

The current knowledge regarding PM in Sinaloa, Mexico is in its early stages.

Reported examples include Podosphaera xanthii (Castagne) U. Braun and Shishkoff in

cucurbits and husk tomatoes (Physalis philadelphica Lam.), Pseudoidium anacardii (F.

Noack) U. Braun and R. T. A. Cook on mango, Erysiphe diffusa (Cooke and Peck) U. Braun

and S. Takam. on common bean, and Podosphaera pannosa (Wallr.) de Bary on roses (Félix-

Gastélum et al. 2016). In addition, Golovinomyces spadiceus (Berk. and M.A. Curtis) U.

Braun was reported in wild sunflower (Félix-Gastélum et al. 2019). In recent years,

Phyllactinia (Ovulariopsis cf. insolita) was reported on Funastrum clausum (Jacq.) Schltr.

and F. cynanchoides (Decne.) Schltr. (Bojórquez-Ramos et al. 2014).

Vallesia glabra (Cav.) Link (Apocynaceae) is a perennial shrub that grows in tropical

and subtropical areas of the Americas (Castañeda-Montoya 2018), including 22 of the 32

Mexican states (Juárez-Jaimes et al. 2007). We observed signs of PM on the leaves of this

3 © The Author(s) or their Institution(s) Botany Page 4 of 24 species in the municipalities of Navolato, Guasave, Ahome and El Fuerte in Sinaloa, Mexico, from February through April, 2020. During sampling collection, we observed that PM was most severe in the lower part of the canopy, mainly in shaded plants growing under perennial trees such as poplar (Populus dimorpha T. S. Brandeg), Manila tamarind (Pithecellobium dulce (Roxb.) Benth), and flame tree (Delonix regia (Bojer ex Hook.) Raf).

The initial signs of the disease consist of small white patches (0.5-1.5 mm in diameter), but as the disease progresses it covers the total abaxial surface of the leaf (Figures

2B and 2C). No signs of the pathogen were observed on the adaxial surface of the leaves.

Abundant structures of the anamorph of the pathogen were observed on infected leaves, however the teleomorph was not observed during the sampling period. Preliminary microscopy observations of the asexualDraft structures of the powdery mildew indicated that the fungus associated with the PM disease belongs to the genus Ovulariopsis, an anamorph of the genus Phyllactinia (Havrylenko et al. 2006; Bojórquez-Ramos et al. 2014).

Seven hundred species of deciduous trees belonging to 69 families distributed mainly in the temperate regions of the northern hemisphere have been found to be affected by PM, in which species of Phyllactinia have been implicated as the causal agent of PM (Amano

1986).

In Argentina, P. chubutiana Harvryl, Takam. and U. Braun (the teleomorph of O. insolita (U. Braun, Kiehr and Delhey) Havryl, S. Takam. and U. Braun) was reported on

Lycium chilense Miers ex Bertero. To identify this fungal species, morphological features of both the teleomorph and the anamorph were examined and molecular techniques were used

(Havrylenko et al. 2006; Braun and Cook 2012). Additionally, Phyllactinia (Ovulariopsis cf. insolita) was reported for the first time on F. clausum and F. cynanchoides in Mexico; since

4 © The Author(s) or their Institution(s) Page 5 of 24 Botany

the teleomorph of the fungus was not observed, the identification included morphological

features of the anamorph and molecular techniques (Bojórquez-Ramos et al. 2014). The

objective of the present study was to identify the fungus associated with PM on V. glabra in

Mexico. To the best of our knowledge, there are no other reports on the incidence of PM

disease on this host plant in Mexico, or anywhere else in the world.

Materials and methods

Sample collection

Ten samples of V. glabra leaves with signs of PM were collected from February 10 through April 15, 2020 in the coastal regionsDraft of the Ahome, El Fuerte, Guasave and Navolato municipalities in Sinaloa, Mexico from 7 to 15 masl (Table 1). There was no rain during the

sampling periods, and the average temperature varied from 13 to 29°C. Samples were placed

in an ice chest at 8-10°C and taken to the laboratory and processed within 48 h after

collection. A representative symptomatic sample was deposited at the herbarium of the

Universidad Autónoma de Occidente in Los Mochis, Sinaloa, Mexico.

Light microscopy and scanning electron microscopy of asexual structures

Specimens were collected from the abaxial leaf surfaces of samples to be used in the

morphometric studies. Slide mounts of mycelia, conidiophores and conidia were prepared by

touching the whitish lesions with clear adhesive tape, and then placing the tape in a drop of

lactophenol-cotton-blue dye on a microscope slide, followed by measuring at 400X

magnification with the aid of an ocular and stage micrometer in a compound microscope

(Labomed; Labo America, Inc., USA). The hyphal width, distance of septum from the

5 © The Author(s) or their Institution(s) Botany Page 6 of 24 branching point of the conidiophore, foot cell length, conidiophore length and width at its midpoint, and the length and width of mature conidia (detached from the conidiophore) were all measured. Forty structures of the anamorph from ten sampling sites (Table 1) were measured (n=400). Images of the structures were obtained using a compound microscope

(Axio Imager M2; Carl Zeiss, Gottingen, Germany) with the same procedure as for the measurements. Micrographs of conidia were taken with a JEOL JSM-53110 LV scanning electron microscope (Bozzola and Russel 1999), after fixing the samples as described by

Ruzin (1999).

Microscopic analysis of infected leaves

Dry infected leaf samples (ca. 0.5 cm) were fixed in 50% ethanol for 24 h at room temperature. Fixed samples were dehydratedDraft through an ethanol series (50, 70, 85, and 100%) for 2 h in each solution. Subsequently, the leaf fragments were transferred to xylol-ethanol

(1:1) for 1 h, 100% xylol for 1 h, and finally embedded in paraffin at 55-60°C during 3 h.

The infiltrated leaves were then placed in small molds to form blocks and sectioned in transverse sections (5 μm) using a microtome (Microm HM 340 E, Thermo Scientific). Slides containing the transverse sections were transferred to xylene in order to remove the paraffin wax. Cross-sections were stained with WGA Alexa Fluor 488 conjugate (Figueroa-López et al. 2014). For observation of the whole tissue, leaf sections were hydrated in 1X PBS supplemented with WGA Alexa Fluor 488 conjugate for 24 h at 4°C. For both the cross- section and whole tissue preparations, confocal microscopy was performed with a laser scanning confocal microscope (Leica TCS SP5 X) using a white laser for 499 nm excitation wavelengths, with emission ranges of 502-548 nm for WGA (green fluorescence) and 598-

706 nm for endogenous fluorescence (red fluorescence).

6 © The Author(s) or their Institution(s) Page 7 of 24 Botany

DNA extraction, PCR, and sequencing

Ten individual specimens from leaves infected with PM (Vg-1 – Vg-10) were

collected using a fine paintbrush for each sample. The samples (30-50 µg) were placed in a

1.6-mL microcentrifuge tube with 800 mL of DNAzol® (Thermo Fisher Scientific;

Cincinnati, OH, USA) for genomic DNA extraction, according to the manufacturer´s

instructions. DNA was resuspended in 30 µL of ultrapure water, then one µL of a ten-fold

dilution was taken as template for the first PCR amplification using the primer set

PHY1/PHY2 (5´- GTTGCTTTGGCGGGCCGG-3´/5´- TTCAGCGGGTATCCCTACCT-

3´) designed for the Phyllactinia group in this work, based on an rDNA region of ~ 540 bp

(Figure 1). One µL of the first PCR reaction was used for a second amplification with the

primer set T4 (5´-TCAACAACGGATCTCTTGGC-3´)Draft (Hirata and Takamatsu 1996) and

PHY2 to amplify a specific region of ~ 375 bp.

PCR mixes were made in a 25.0 µL reaction volume that included: 17.65 µL of

ultrapure water, 2.5 µL of buffer, 0.75 µL of 50 µM MgCl2, 1.0 µL of each primer (10 µM),

1 µL of 10 mM dNTP mix, 0.1 µL (1 U) of Taq DNA polymerase (Invitrogen, USA), and

1.0 µL of template DNA. The PCR amplifications were performed in an Apollo ATC-201

PCR cycler (Nyx Technik, San Diego, California, USA) using the following conditions: an

initial denaturing step at 94ºC for 5 min; subsequently, 35 cycles consisting of 30 s at 94ºC

followed by 30 s at 60ºC for annealing and 30 s at 72ºC for extension; and a final extension

cycle of 5 min at 72ºC. PCR products were purified using the QIAquick PCR purification

Kit (Qiagen®, USA) and sequenced with the PHY2 primer in an ABI 3730xl sequencer

(Applied Biosystems, Foster City, CA, USA) at the National Laboratory of Genomics for

Biodiversity (Langebio) in Irapuato, Mexico.

7 © The Author(s) or their Institution(s) Botany Page 8 of 24

Phylogenetic analysis

Sequences were edited in BioEdit version 7.0.5.3. (Hall 1999) and compared with sequences in the NCBI (National Center for Biotechnology Information) using the BLAST-

N software and the Megablast algorithm. MEGA X (Kumar et al. 2018) was used for alignment and phylogenetic analysis. All sequences were deposited in GenBank under the accession numbers MT909512-MT909521 (Table 1). Sequences were aligned with 26 reference sequences from the Phyllactinia spp. and a sequence of Leveillula verbasci (Jacz.)

Golovin as an outgroup (Saenz and Taylor 1999; Khodaparast et al. 2002; Takamatsu et al.

2008; Khodaparast et al. 2016), using the MUSCLE alignment program (Edgar 2004) implemented in MEGA X. The Akaike information criteria (AIC) were used for substitution model selection. A phylogenetic treeDraft was constructed using the general time reversible (GTR) model and the maximum likelihood (ML) method. Among-site rate variation was modelled by a gamma distribution (four categories). Tree topology support was assessed by

1,000 bootstrap replicates.

Results

Morphological characteristics

Since the teleomorph of the fungus associated with the disease was not observed during the sampling period, we examined the morphological characteristics of the anamorph.

Mycelia occurred only on the abaxial surface of the leaves. They were dense and persistent, and predominantly ectophytic but also endophytic, forming patches with a powdery appearance (Figure 2B) that expanded and covered the whole abaxial surface of the leaf

(Figure 2C). Hyphae were straight, 2.5-7.5 µm in width (5.0 µm on average) with a wavy

8 © The Author(s) or their Institution(s) Page 9 of 24 Botany

appearance (Figure 2E) primarily near the conidiophores. Hyphal appressoria were nipple-

shaped, bifurcated or irregularly branched, and solitary or present in opposite pairs (Figure

2D). The distance of the basal septum from the conidiophore above the junction with the

mother cell was highly variable (7-18 µm, 12 µm on average) (Figure 2E); in some cases,

there was no septum in the conidiophore. Conidiophores were simple, with a verrucose outer

wall (Figure 2G) produced from external hyphae, and foot cells measuring (6.2-) 30-50 (-

82.5) in length x (4.5-) 6-8 µm in width (42.3 x 6.7 µm on average). Conidiophores were

(30-) 60-87.5 (-130) in length x (3.7-) 5.0-7.5 (-8.7) µm in width (77.0 x 6.4 µm on average),

with (0-) 1-3 (-5) cells following the basal septum of the conidiophore (Table 2). The

immature conidia attached on the apex of conidiophores were ellipsoid to lanceolate in shape,

and displayed abundant granulation (Figure 2G). In contrast, mature conidia (detached from

the conidiophore) were constricted atDraft the midpoint and were rectangular or ellipsoid in shape

with reticular wrinkling and granulate outer walls (Figures 2F and 2H) measuring (32-) 42-

47 (-70) x (12-) 15-20 (-27) µm (45.4 x 18.4 µm on overage). The average length:width ratio

was 2.5 (Table 2), and mature conidia exhibited 5-6 prominent sub-terminal or sub-apical

protuberances (Figure 2H).

An abundance of mycelia on the abaxial surface of leaves was confirmed by confocal

microscopy (data not shown). Some hyphae branched and penetrated the leaves through the

stomata and reached the intercellular leaf spaces (Figure 3A, green color). The hyphae were

located in the spongy mesophyll cells and palisade mesophyll cells, although haustoria

showing an irregular shape were only observed in the spongy mesophyll cells (Figure 3B,

white arrows). Hyphae were not found on the adaxial leaf surface.

Phylogenetic analysis

9 © The Author(s) or their Institution(s) Botany Page 10 of 24

The partial ITS sequences from ten specimens were compared with ITS data in the

NCBI database. The results revealed a 99.34% identity with ITS sequences retrieved from specimens of P. chubutiana deposited in GenBank with the accession numbers KC122682

(Bojórquez-Ramos et al. 2014) and AB243690 (Havrylenko et al. 2006). The phylogenetic analysis showed the grouping of all V. glabra specimens with P. chubutiana, in a well- supported clade (99% bootstrap; Figure 4), confirming their identity.

Discussion

Our results demonstrate that the anamorph of the powdery mildew fungus associated with V. glabra is morphologically similar to the genus Ovulariopsis Pat. and Har. (Braun and

Cook 2012). The hyphal appressoria produced by the fungus were similar to those reported in Mexico for Phyllactinia (OvulariopsisDraft cf. insolita) on F. clausum and F. cynanchoides (Bojórquez-Ramos et al. 2014). Observations by light and scanning electron microscopy revealed that the fungus produces lanceolate conidia when immature, and cylindrical ones when mature (Havrylenko et al. 2006). The protuberant conidia and the structure of the conidiophores coincide with those of P. chubutiana (Havrylenko et al. 2006) and Phyllactinia

(Ovulariopsis cf. insolita) (Bojórquez-Ramos et al. 2014). The verrucose conidiophores and conidia of O. insolita on V. glabra agree with previous observations of O. insolita on F. clausum and F. cynanchoides in Mexico (Bojórquez-Ramos et al. 2014), but differ from P. chubutiana (an anamorph of Ovulariopsis insolita) on Lycium chilense, in which this characteristic is not reported (Havrylenko et al. 2006; Braun and Cook 2012). Although this species may infect the adaxial and abaxial parts of the leaves of L. chilense (Havrylenko et al. 2006), F. clausum and F. cynanchoides (Bojórquez-Ramos et al. 2014), in V. glabra we only found the fungus on the abaxial leaf surface. It is important to note that the incidence and severity in this host plant are higher in shaded plants than in other PM species (Yarwood

10 © The Author(s) or their Institution(s) Page 11 of 24 Botany

1973). The asexual structures of our specimens shared morphometric similarities with those

of Phyllactinia (Ovulariopsis cf. insolita) on Funastrum spp. in Mexico. However, the

number of cells above the foot cell of the conidiophore in Ovulariopsis found on V. glabra

reached up to five cells, in contrast to the PM on Funastrum spp. with a maximum of three

cells (Table 2).

Many species of powdery mildews have a high degree of host specialization, infecting

only one or several closely related host genera (Ridout 2009). To date, it is not known

whether the PM species found in three species (F. clausum, F. cynanchoides and V. glabra)

of the family Apocynaceae in Sinaloa can infect other members of this family. Considering

that this family includes 385 species widely distributed across Mexico (Juárez-Jaimes et al.

2007), this topic should be considered in future research.

Our molecular studies confirmDraft that the PM on V. glabra corresponds to Phyllactinia

chubutiana and its asexual morph, O. insolita, found in Argentina on the Solanaceous plant

L. chilense (Havrylenko et al. 2006), as well as Phyllactinia (Ovulariopsis cf. insolita), which

was previously found on F. clausum and F. cynanchoides in Mexico (Bojórquez-Ramos et

al. 2014). Therefore, the described PM species on V. glabra is named Phyllactinia

chubutiana, nom. cons. (= Oidium insolitum, Ovulariopsis insolita), the teleomorph of the

fungus is still required in order to confirm that it corresponds to the same species.

Phyllactinia species are usually host-specific, i.e., they are confined to hosts of single

genera or of a few allied genera belonging to a particular pant family (Takamatsu et al. 2008;

Braun and Cook 2012). There are only few exceptions, such as P. fraxini that can also occur

on Asclepias spp. (Asclepiadaceae) and Wisteria sinensis (Fabaceae) (Braun and Cook 2012).

P. chubutiana is a further example for a Phyllactinia species with wider host range,

exceeding the limits of a single plant family

11 © The Author(s) or their Institution(s) Botany Page 12 of 24

Currently, information about the distribution of P. chubutiana around the world is limited. While this is the second report in Mexico, and the third world-wide, it is the first report in the host plant V. glabra. Due to the large number of species in the family

Apocynaceae, new research directions should focus on the determination of PM species that occur in other members of this family.

Acknowledgments

The authors thank Dr. Jerzy Rzedowski Rotter (Emeritus professor at the Ecology Institute,

Mexico) for the identification of the species Vallesia glabra included in the present study and Uwe Braun (Full Professor at the Martin Luther University, Halle-Wittenberg Institute of Biology, Germany) for his valuable revision of the manuscript, as well as the graduate division of the Universidad AutonomaDraft de Occidente for financial support through the PIFIP-

2019. The authors are grateful to the National Council on Science and Technology

(CONACyT) for financial support from CONACyT grant no. 250738. We thank Dr. Brandon

Loveall from Improvence editing services for English proofreading of the manuscript.

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Ainsworth, F. K. Sparrow, and A. S. Sussman. Academic Press. New York, USA. pp.

71-86.

Draft

Table 1. GenBank accession numbers for the sequencing data from Phyllactinia chubutiana (anamorph of Ovulariopsis insolita) obtained in

this study.

Sample Locality Collection date Coordinates GenBank accession no.

Vg-1 Country Club, February 10, 25°47'25''N MT909512 Ahome 2020 109°00'28''W Vg-2 El Portón, February 10, 25°47'23''N MT909513 Ahome 2020 109°02'23''W Vg-3 Plan de San February 21, 25°46'23''N MT909514 Luis, Ahome 2020 109°03'42''W Vg-4 Guasave, norte March 2, 2020 25°35'12''N DraftMT909515 108°29'15''W Vg-5 Guasave II, sur March 2, 2020 25°28'43''N MT909516 108°34'54''W Vg-6 Charay, El March 9,2020 25°59'32''N MT909517 Fuerte 108°51'28''W Vg-7 Pochotal, El March 9,2020 25°59'43''N MT909518 Fuerte 107°42'21''W Vg-8 Caimancito, April 2, 2020 24°48'43''N MT909519 centro, 107°42'21''W Navolato, Vg-9 Caimancito II, April 2, 2020 24°49'10''N MT909520 norte 107°39'23''W Navolato Vg-10 Los Suárez, April 15, 2020 25°56'57''N MT909521 Ahome 109°11'20''W

Table 2. Comparison of the morphometric characteristics of the anamorph Ovulariopsis insolita on Vallesia glabra with the same anamorph

on three different host plants.

Species/place of collection/measurements (µm)

P. chubutiana Phyllactinia (Ovulariopsis cf. Phyllactinia (Ovulariopsis cf. insolita) Argentina (anamorph of insolita) Mexico Ovulariopsis insolita) Mexico Host plant(s) Lycium chilense (Solanaceae) Funastrum clausum and Vallesia glabra (Apocynaceae) F. cynanchoides (Apocynaceae) Hyphae width 2.5-8.0 3-9 (5.5 on average) 2.5-7.5 (5.0 on average) Foot cell of conidiophore 6-49 x 5-15 Draft10-74(-83) x (6.2-) 30-50 (-82.5) x lengthwidth (4-) 5-10 (-11) (39 x 8 on (4.5-) 6-8 (42.3 x 6.7 on average) average) Conidiophore length -.- -.- (30-) 60-87.5 (-130) x (3.7-) 5.0-7.5 (-8.7) (77.0 x 6.4 on average) No. of cells following foot cell in -.- (0-)1-2 (3) (0-) 1-3 (-5) conidiophore Conidia lengthwidth 27-52 x 12-22 (34-) 38-54 (- 56) x (32.5-) 42.5-47.3 (-70) x (14-) 16-23 (-24) (12.5-) 15-20 (-27.5) (45.4 x 18.4 on average) Conidia length/width ratio -.- 2.2 2.5 Subterminal protuberance 4-8 5-6 5-6 Position of the basal septum in -.- 2-15 5-18 (12.5 on overage) the mother cell of conidiophore Granular appearance of -.- Positive Positive conidiophore in conidium References Havrylenko et al. 2006 Bojórquez-Ramos et al. 2014 The present study -.- = Not reported

Figure legends

Figure 1. Schematic diagram showing the annealing sites of the newly designed PCR primers

PHY1 and PHY2, as well as the T4 primer (Hirata and Takamatsu 1996), in the ribosomal

DNA region. The arrows indicate the direction of amplification.

Figure 2. Signs of powdery mildew Phyllactinia chubutiana, nom. cons. (= Oidium

insolitum, Ovulariopsis insolita; specimen Vg-1) on leaves of Vallesia glabra, and

morphological characteristics of the pathogen. (A) Healthy leaves of the host plant; (B) white

patches of powdery mildew on the abaxial part of the leaves; (C) abaxial part of the leaves

covered by powdery mildew; (D) light micrograph showing a hypha with appressorium (black arrow); (E) conidiophores withDraft basal septum above the mother cell (black arrow); (F) mature detached conidia indicated by arrows; (G) scanning electron micrograph of immature

conidia on a conidiophore; (H) detached turgid mature rectangular conidium slightly

constricted at the midpoint showing netted wrinkling and a granulose outer wall as well as

subterminal protuberances. Both examples display granular ornamentation. Scale bars: D-F,

20 µm; G, 10 µm; H, 1 µm.

Figure 3. Phyllactinia infects Vallesia glabra (specimen Vg-1) through its stomata openings.

A) Visualization of the Phyllactinia hypha (green fluorescence) through the stomatal

opening. B) Cross-section of a V. glabra leaf (which emits endogenous red fluorescence)

colonized by Phyllactinia (green fluorescence). WGA Alexa-488 was used to detect fungal

hyphae in the colonized leaf.

Figure 4. A) Maximum likelihood tree based on the ITS region of Phyllactinia specimens.

The tree was constructed in MEGA X (bootstrap = 1000), using the general time reversible

(GTR) model; four gamma categories (G) and invariant sites (I) were used to model the among-site rate variation. The corresponding sequence of Leveillula verbasci was used as an outgroup. B) Detailed view of the collapsed node in panel A, showing the grouping of

Vallesia glabra isolates (in bold) with P. chubutiana. Database accession numbers of the sequences are provided in parentheses. Bootstrap values are given as percentages. The scale bar indicates the number of nucleotides per site.

Draft

Figure 1. Schematic diagram showing the annealing sites of the newly designed PCR primers PHY1 and PHY2, as well as the T4 primer (Hirata and Takamatsu 1996), in the ribosomal DNA region. The arrows indicate the direction of amplification

147x30mm (300 x 300 DPI)

Draft

Figure 2. Signs of powdery mildew Phyllactinia chubutiana, nom. cons. (= Oidium insolitum, Ovulariopsis insolita; specimen Vg-1) on leaves of Vallesia glabra, and morphological characteristics of the pathogen. (A) Healthy leaves of the host plant; (B) white patchesDraft of powdery mildew on the abaxial part of the leaves; (C) abaxial part of the leaves covered by powdery mildew; (D) light micrograph showing a hypha with appressorium (white arrow); (E) conidiophores with basal septum above the mother cell (white arrow); (F) mature detached conidia indicated by arrows; (G) scanning electron micrograph of immature conidia on a conidiophore; (H) detached turgid mature rectangular conidium slightly constricted at the midpoint showing netted wrinkling and a granulose outer wall as well as subterminal protuberances. Both examples display granular ornamentation. Scale bars: D-F, 20 µm; G, 10 µm; H, 1 µm.

254x144mm (138 x 131 DPI)

Figure 3. Phyllactinia infects Vallesia glabra (specimen Vg-1) through its stomata openings. A) Visualization of the Phyllactinia hypha (green fluorescence) through the stomatal opening. B) Cross-section of a V. glabra leaf (which emits endogenous red fluorescence) colonized by Phyllactinia (green fluorescence). WGA Alexa- 488 was used to detectDraft fungal hyphae in the colonized leaf. 176x87mm (300 x 300 DPI)

Figure 4. A) Maximum likelihood tree based on the ITS region of Phyllactinia specimens. The tree was constructed in MEGA X (bootstrap = 1000),Draft using the general time reversible (GTR) model; four gamma categories (G) and invariant sites (I) were used to model the among-site rate variation. The corresponding sequence of Leveillula verbasci was used as an outgroup. B) Detailed view of the collapsed node in panel A, showing the grouping of Vallesia glabra isolates (in bold) with P. chubutiana. Database accession numbers of the sequences are provided in parentheses. Bootstrap values are given as percentages. The scale bar indicates the number of nucleotides per site.

312x182mm (300 x 300 DPI)