Article Induced Volatile Emissions, Photosynthetic Characteristics, and Pigment Content in Leaves Infected with the Erineum-Forming erinea

Corina Popitanu 1,†, Andreea Lupitu 2,† , Lucian Copolovici 2,* , Simona Bungău 1 , Ülo Niinemets 3,4 and Dana Maria Copolovici 2

1 Biomedical Sciences Doctoral School, University of Oradea, University St. no. 1, 410087 Oradea, Romania; [email protected] (C.P.); [email protected] (S.B.) 2 Institute for Research, Development and Innovation in Technical and Natural Sciences, Faculty of Food Engineering, Tourism and Environmental Protection, Aurel Vlaicu University of Arad, Elena Drăgoi St., no. 2, 310330 Arad, Romania; [email protected] (A.L.); [email protected] (D.M.C.) 3 Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, 51006 Tartu, Estonia; [email protected] 4 Estonian Academy of Sciences, Kohtu 6, 10130 Tallinn, Estonia * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: Persian walnut (Juglans regia L., Juglandaceae), one of the essential nut crops, is affected by different diseases, including mite attacks which result in and erineum formation. As the   proportion of leaf area covered by mite or erineum is typically relatively low, the impact on tree photosynthetic productivity is often considered minor, and no pest control management is Citation: Popitanu, C.; Lupitu, A.; usually suggested. However, the effect of erineum-forming on walnut photosynthesis might be Copolovici, L.; Bung˘au,S.; Niinemets, disproportionately larger than can be predicted from the leaf area impacted. In the present study, we Ü.; Copolovici, D.M. Induced Volatile studied how the foliage photosynthetic characteristics, pigment contents, and stress-induced volatile Emissions, Photosynthetic organic compounds scaled with the severity of varied from 0% (control trees) to 9.9%, by Characteristics, and Pigment Content in Juglans regia Leaves Infected with erineum-forming mite Aceria erinea in J. regia. Both leaf net assimilation rate (up to 75% reduction) the Erineum-Forming Mite Aceria and stomatal conductance (up to 82%) decreased disproportionately, increasing infection severity. erinea. Forests 2021, 12, 920. Leaf total chlorophyll and β-carotene contents also decreased with infection severity, although https://doi.org/10.3390/f12070920 the reduction was less than for photosynthetic characteristics (28% for chlorophyll and 25% for β-carotene). The infection induced significant emissions of green leaves volatiles ((Z)-3-hexenol, Academic Editor: Luís González (E)-2-hexenal, (Z)-3-hexenyl acetate and 1-hexanol), monoterpenes and the homoterpene 3-(E)-4,8- dimethyl-1,3,7-nonatriene, and these emissions scaled positively with the percentage of leaf area Received: 8 June 2021 infected. These results collectively indicate that erineum-forming mite infection of walnut leaves Accepted: 13 July 2021 results in profound modifications in foliage physiological characteristics that can significantly impact Published: 15 July 2021 tree photosynthetic productivity.

Publisher’s Note: MDPI stays neutral Keywords: green leaf volatiles; induced emissions; monoterpene emission; photosynthesis; quantita- with regard to jurisdictional claims in tive responses; volatile organic compounds published maps and institutional affil- iations.

1. Introduction Persian walnut (Juglans regia L., Juglandaceae) is a deciduous tree native to Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. central Asia. It is one of the most important nut crops with a wide area of cultivation, This article is an open access article including Europe, North America, Central, and East Asia, North Africa, New Zealand [1]. distributed under the terms and In Europe, it is primarily grown in central to southern countries, although cultivars selected conditions of the Creative Commons for superior cold hardiness can survive in the south part of Northern Europe [2]. The most Attribution (CC BY) license (https:// extensive J. regia plantations are in Turkey, France, Romania, Serbia, and Hungary. As a tree creativecommons.org/licenses/by/ species with a sequenced genome, it becomes one of the model species for physiological 4.0/). studies in woody plants [3].

Forests 2021, 12, 920. https://doi.org/10.3390/f12070920 https://www.mdpi.com/journal/forests Forests 2021, 12, 920 2 of 12

Juglans regia is an early-successional very light-demanding species growing best in deep fertile soils [4]. As a widely planted species, J. regia is confronted by a broad range of pests, including fungi, bacteria, viruses, and mite , and parasites that could disturb roots, fruits, stems, or leaves [1,2,5]. The J. regia leaves can be attacked by a diversity of mites and , including leaf miners, chewing and piercing insect herbivores [6–8], and gall- and erineum-forming mites [9–11] that can lead to extensive defoliation and premature leaf senescence and reduction in leaf physiological activity, thereby potentially having significant economic impacts [5]. However, mite gall and erineum formation infrequent in J. regia plantations [9–11], the physiological effects of gall and erineum formation have not been characterized in this species. Gall and erineum formation result from an active growth reaction of cell plants in reaction to the insect or mite attacks, leading to the creation of tumor-like structures that provide shelter and food for the occupants [12,13]. The gall-inducing insects are found in the orders , Thysanoptera, Diptera, gall- and erineum-forming mites in (superfamily Eriophyoidea)[14]. Individual eriophyid species are highly specialized plant feeders affecting only a very narrow range of hosts. Besides high host specificity, a very high diversity of eriophyoid mites (more than 1000 species) can induce galls and erinea on plant species from a wide range of families [15,16]. As gall and erineum formation does not result in defoliation and the surface area covered by galls and erinea is generally a relatively small fraction of the total area, the impact of leaf galling on foliage physiological activity might be considered small. However, available evidence indicates that leaf infection by gall- and erineum-inducing parasites can result in disproportionately greater physiology and biochemistry alterations than predicted based on the percentage of the infected area [17]. In particular, infestation by gall-forming typically leads to strongly reduced leaf photosynthetic pigment content as observed in Populus nigra var. italica leaves by infestion with petiole gall (Pemphi- gus spyrothecae)[18], and Alstonia scholaris leaves plagued by Pauropsylla tuberculata [19]. Typically, the gall or erineum-induced leaf physiological alterations include reductions in both light-saturated stomatal conductance and net assimilation rate as confirmed for gall-forming cynipid wasp in Quercus robur [17] and eriophyoid erineum-and gall-forming mite infections in cordata and [20]. Similarly, both stomatal conductance and net assimilation rate were reduced in Carya illinoensis leaves infested by Phylloxera notabilis galls [21]. A lower photosystem II efficiency was found in Carya glabra infested with cecidomyiid midge galls [22]. Net assimilation rate, stomatal conductance, and photosystem II activity were reduced in the leaves of Machilus thunbergii galled by Daphnephila taiwanensis [23]. However, depending on the type of gall inducer, leaf CO2 and H2O exchange can be disturbed to a different extent, potentially leading to modifi- cations in leaf water use efficiency [17,20,24]. For example, in leaves of P. × petrovskiana infested with P. spyrothecae net assimilation rate per area decreased by almost five-fold likely due to restricted nitrogen and carbon availability for leaf lamina assembly. At the same time, increasing the infestation severity determinea the stomatal conductance in- crease [25]. Furthermore, net assimilation rate and stomatal conductance even increased in Silphium integrifolium leaves infested with the apical meristem galler Antistrophus silphii [26], suggesting profound modifications in sink–source relationships induced by the galler. Apart from photosynthetic modifications, to respond to feeding or fungal infection, plants emit various volatiles induced by stress, such as monoterpenes, sesquiter- penes, and green leaf volatiles (GLV) [27–31]. These compounds are implicated in at- mospherical chemical reactions with pollutants (as NOx) and determine tropospheric ozone concentration increasing [32]. Much fewer studies have looked at volatile emissions from plants infected with -formed galls or erinea. Leaves infected with gall- or erineum-forming arthropods typically have enhanced emissions of GLV [17,20], suggesting a sustained damage resulting from the feeding of arthropods. However, induced and constitutive emissions might be differently affected by different gall- or erineum-forming organisms. In [25], both constitutive isoprene and induced monoterpene emissions scaled Forests 2021, 12, 920 3 of 12

positively with the infestation severity by petiole gall in leaves of P. × petrovskiana. In [17], in -infected leaves of Quercus robur, constitutive isoprene emissions de- creased in all cases with increasing infection severity. In contrast, the emission of monoter- penes was increased in leaves infected by albipes, Cynips divisa, and C. quercusfolii wasps and reduced in leaves infected by N. anthracinus [17]. In another study, monoterpene emissions were greater in leaves of Tilia cordata infected by tiliae gall-forming and E. exilis erineum-forming mites, while monoterpene emissions decreased in Alnus glutinosa leaves infected by E. inangulis gall-forming mites [20]. These studies provide encouraging evidence that alterations in constitutive and induced emissions might be used to diagnose the type and severity of infections of gall- and erineum-forming arthropods. In J. regia, the gall- and erineum-forming mites all belong to Eriophyoidea: Aceria brachytarsus, A. avanensis, A. erinea, and A. tristata [10]. Walnut blister mite (Aceria erinea Nalepa) is a mite that causes erinea resembling yellowish blisters on the upper surface on walnut leaves [11,33]. The blisters correspond to hollows on the leaf underside; the hollows are covered with a dense whitish or pale brown mat of hypertrophic trichomes harboring mites [34] for a description of erineum structure. Although having a spectacular visual appearance, the A. erinea-induced erinea typically covers a low proportion of leaf area, less than 10%. Still, it is likely that leaf physiological characteristics are altered by erinea to a much greater degree than expected based on the degree of infection. In this study, we hypothesized that A. erinea infections (1) reduce photosynthesis char- acteristics and photosynthetic pigment concentration, and elicit volatile stress emissions in leaves of J. regia in an infection severity-dependent manner, and (2) that the relative alterations in leaf traits are greater than changes in the proportion of area infected. This study demonstrates that even moderate infections of A. erinea signals strongly reduced leaf photosynthetic activity and enhanced production of stress volatiles that can be employed to predict the reduction in foliage photosynthetic productivity.

2. Materials and Methods 2.1. Study Site and Plant Material The study takes place in Arad, Romania (46◦170 N, 21◦170 E, elevation ~60 m above sea level) close to Mures river in the middle of July 2020. For July 2020, the average minimum air temperature was 14.6 ◦C, and the maximum air temperature was 27.8 ◦C (National Meteorological Administration. Available online: https://www.inmh.ro (accesssed on 28 June 2020)). The Juglans regia trees chosen for performing the measurements were 20–30 years old and 20–25 m tall. South-exposed leaves with varying degrees of visual in- fection symptoms were selected to measure gas-exchange parameters, and volatile organic compound (VOC) emission rates with the leaves attach to the tree. Control non-infected leaves were measured in nearby healthy trees. After that, all leaves have been collected, used to assess the degree of infection followed by extraction of pigments.

2.2. Foliage Gas-Exchange Measurements In the field, photosynthetic characteristics of attached leaves with different degrees of infection were measured using a portable gas-exchange system GFS-3000 with 8 cm2 leaf chamber (Waltz, Effeltrich, Germany) as described in [35] under the following environ- −1 mental conditions: 65% air humidity in the chamber, 400 µmol mol CO2 concentration, 1000 µmol m−2 s−1 (10% blue and 90% red LED light) the incident light intensity, and 25 ◦C leaf temperature. After the chamber’s enclosure, the leaf was maintained until stomata opened and steady-state CO2 and water vapor exchange rates were reached. The stabilization period was typically 20–30 min. Once in the steady-state, foliage gas exchange rates were logged. For calculation of the values of stomatal conductance to water vapor (gs) and net assimilation (A) the same procedure described in [36] has been used. In representative leaves with two different degrees of infection, leaf gas exchange’s parameters light-response curves were also recorded with the same system (GFS-3000, Forests 2021, 12, x FOR PEER REVIEW 4 of 12

Forests 2021, 12, 920 4 of 12

In representative leaves with two different degrees of infection, leaf gas exchange’s parameters light-response curves were also recorded with the same system (GFS-3000, HeinzHeinz WalzWalz GmbH, GmbH, Effeltrich, Effeltrich, Germany). Germany). TheThe light-response light-responsecurve curve measurements measurementswere were −2 −1 concededconceded inin the the following following sequence sequence (light (light intensities intensities in inµ µmolmol m m−2 s−1):): 00 → 50 → 100100 → 200200 → 300300  400→ 400  500→ 500 600→ 600 800→ 800 1000→ 1000 1200→  12001500 → 1800.1500 → 1800.Other environmental conditions were maintained as above. At each light intensity, the gasOther exchange environmental rates were conditions stabilized were until maintained the steady-state as above. values At were each observed. light intensity, Light- theresponse gas exchange curves rateswere weremeasured stabilized in triplicate until the for steady-state control and values leaves were with observed. approximately Light- response3.5% and curves 5.1% infection. were measured The light in triplicate response for curves control were and fitted leaves by with the approximately rectangular hyper- 3.5% and 5.1% infection. The light response curves were fitted by the rectangular hyperbola of bola of Smith [37] as in [38], and dark respiration rate (Rd), initial quantum yield (α) and Smith [37] as in [38], and dark respiration rate (R ), initial quantum yield (α) and maximum maximum net assimilation rate (Amax) were obtained.d net assimilation rate (Amax) were obtained. 2.3. Leaf Pigment Analysis 2.3. Leaf Pigment Analysis Pigment extraction was accomplished according to the method defined in [39], fol- Pigment extraction was accomplished according to the method defined in [39], fol- lowed by the HPLC analysis described in [40]. lowed by the HPLC analysis described in [40].

2.4.2.4. VolatileVolatile SamplingSampling andand GC–MSGC–MS AnalysesAnalyses AnAn airair samplesample pumppump 210-1003210-1003 MTX MTX (SKC (SKC Inc., Inc., Houston, Houston, TX, TX, USA) USA) with with the the constant constant flowflow waswas usedused to sample volatile volatile organic organic compounds compounds (VOC) (VOC) via via the the outlet outlet of ofthe the gas gas--ex- exchangechange system system using using the the same same procedure procedure and and multibed multibed stainless stainless steel steel cartridge cartridge as de- as describedscribed in in [41] [41.]. The The flow flow rate rate for for the volatile collectioncollection waswas200 200 mL mL min min−−11,, andand volatilesvolatiles fromfrom thethe 44 L chamber air air was was collected. collected. The The adsorbent adsorbent cartridges cartridges were were analyzed analyzed for fordif- differentferent volatile volatile organic organic compounds compounds as green as green leaf leafvolatiles volatiles (GLV) (GLV) and terpenes and terpenes using using a Shi- amadzu Shimadzu TD20 TD20 automated automated cartridge cartridge desorber desorber integrated integrated with witha Shimadzu a Shimadzu 2010 2010 Plus PlusGC– GC–MSMS instrument instrument (Shimadzu (Shimadzu Corporation, Corporation, Kyoto, Kyoto, Japan) Japan) following following the the method method of [[41,42]41,42].. VolatileVolatile emission emission rates rates were were calculated calculated according according to to Niinemets Niinemets et et al. al. [43 [43]]

2.5.2.5. EstimationsEstimations ofof thethe LeafLeaf AreaArea andand thethe DegreeDegree ofof Infection Infection TheThe leavesleaves were scanned at at 200 200 dpi, dpi, and and the the leaf leaf area area was was infected. infected. The The projected projected leaf leafarea area was wasestimated estimated using using custom custom-built-built software software “Leaf “Leaf Area AreaMeasurement” Measurement” (www.plant (www.- plant-image-analysis.orgimage-analysis.org, accessed, accessed on 5 onMay 5 May2021) 2021) (Figure (Figure 1 for1 representativefor representative examples examples of con- of controltrol and and infected infected leaflet leaflets).s).

FigureFigure 1.1. Representative photos photos of of JuglansJuglans regia regia leafletsleaflets without without (control) (control) and and with with the the erineum erineum-- forming mite Aceria erinea infection. forming mite Aceria erinea infection.

2.6.2.6. StatisticalStatistical AnalysisAnalysis andand DataData HandlingHandling AltogetherAltogether 2020 leavesleaves withwith differentdifferent degreesdegrees ofof infection infection andand fivefive control control leaves leaves were were measuredmeasured for for foliage foliage gas-exchange gas-exchange characteristics characteristics and and volatile volatile emissions. emissions. Initially, Initially, we we tested them using the null hypothesis (parametric one-way ANOVA) whether there were any differences between different tree leaves. Because there was no difference and it

Forests 2021, 12, x FOR PEER REVIEW 5 of 12

Altogether 20 leaves with different degrees of infection and five control leaves were Forests 2021, 12, 920 measured for foliage gas-exchange characteristics and volatile emissions. Initially,5 of we 12 tested them using the null hypothesis (parametric one-way ANOVA) whether there were any differences between different tree leaves. Because there was no difference and it was demonstrated that the tree effect was not significant, all data were pooled for the regres- sionwas demonstratedanalyses. Linear that and the non tree-linear effect regressions was not significant, (Michaelis all-Menten data were type pooled equation: for y the = aregressionx/(b + x)) analyses.were used Linear to explore and non-linearthe relationships regressions of foliage (Michaelis-Menten physiological characteristics type equation: · andy = apigmentx/(b + x))contentswere used with to the explore degree the of relationshipsinfection. The of parameters foliage physiological of the light characteris- response curvestics and among pigment control contents and with differently the degree-infected of infection. leaves The(n = parameters3 in each case) of the were light compared response curves among control and differently-infected leaves (n = 3 in each case) were compared by by parametric ANOVA followed by the Tukey post hoc test for pairwise comparison. All parametric ANOVA followed by the Tukey post hoc test for pairwise comparison. All statis- statistical analyses were conducted with GraphPad Prism version 9.1.2 for Windows tical analyses were conducted with GraphPad Prism version 9.1.2 for Windows (GraphPad (GraphPad Software, San Diego, Ca, USA), and the statistical tests were considered sig- Software, San Diego, CA, USA), and the statistical tests were considered significant at nificant at p < 0.05. p < 0.05.

3.3. Results Results 3.1.3.1. Photosynthesis Photosynthesis Characteristics Characteristics of of Leaves Leaves Infected Infected with with A. A. erinea erinea TheThe infection withwith A.A. erinea erineainduced induced a drastica drastic decrease decrease of averageof average net net assimilation assimilation rate ratefrom from 14.8 14.8± 1.0  µ1.0mol µmol m− 2ms−2− 1s−1in in control control leaves leaves to to around around three threeµ molµmol m m−2−2s s−−11 inin heavily infectedinfected leaves leaves (Figure (Figure 22a).a).

Figure 2. LeafLeaf net net CO CO22 assimilationassimilation rate rate ( (aa)) and and stomatal stomatal conductance conductance to to water water vapor vapor ( (bb)) concerning concerning the the percentage percentage of leaf leaf area infected with A. erinea inin J. regia regia.. P Photosynthetichotosynthetic measurements measurements were conducted under the following conditions: 65% 65%    −11 2 −22 −1 1 chamber air humidity, 400400µ µmolmol mol mol CO2 concentration, 1000 µμmolmol mm ss incidentincident light light intensity, intensity, and and leaf leaf temperature temperature of 25 °C◦C( (nn == 25). 25). The The data data have have been been fitted fitted using using non non-linear-linear regression regression y y = = a a·x/(x/(bb + +x). x).

EvenEven a a minor infection infection (0.9% (0.9% degree degree of infection) reduced leaf assimilation rate rate by 110–15%0–15% (Figure 22a).a). StomataStomata conductanceconductance toto waterwater vaporvapor declineddeclined withwith thethe infectioninfection raterate fromfrom 151.6 ± 2.3 mmol mm−−22 ss−1− 1inin control control leaves leaves to to values ofof 23–2523–25 mmol mmol m m−−22 ss−−11 inin leaves leaves withwith more than 7% infection (Figure 22b).b).

3.2.3.2. Modification Modification of of Light Light Responses Responses of of Photosynthesis Photosynthesis by A. erinea Infection AnalysisAnalysis of of the the light light response response curves curves further further confirmed confirmed the major the major reduction reduction in photosyn- in pho- thesistosynthesis rate. The rate. maximum The maximum net photosynthetic net photosynthetic rate was higher rate was for control higher plants for control (16.41 plants 0.10 −2 −1 −2 −1 µmol(16.41 m±−2 0.10s−1) thanµmol for m infecteds ) thanplants for (11.94 infected  0.28 plants µmol (11.94m−2 s−1± for0.28 averageµmol degree m s of infectionfor aver- −2 −1 ofage 3.5% degree and of10.66 infection  0.27 µmol of 3.5% m−2 and s−1 for 10.66 5.1%± 0.27infectiµmolon, p m < 0.05s forfor comparison 5.1% infection, with thep < con- 0.05 trol,for comparisonFigure 3a). The with initial the quantum control, Figureyield decreased3a). The from initial 0.0324 quantum  0.0005 yield μmol decreased μmol–1 in from con- −1 −1 trol0.0324 leaves± 0.0005 to 0.0083µmol  0.0003µmol μmolin controlμmol–1 in leaves leaves to with 0.0083 5.1%± infestation0.0003 µmol (p µ< mol0.05). However,in leaves differenceswith 5.1% infestationin the dark res (p

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Figure 3. Representative light (photosynthetic photon flux density, PPFD) responses of net CO2 assimilation rate (a) and Figure 3. Representative light (photosynthetic photon flux density, PPFD) responses of net CO assimilation rate (a) and stomatalFigure 3. conductance Representative to waterlight (photosyntheticvapor (b) for J. photonregia control flux density, (non-infected) PPFD) andresponses A. erinea of -netinfected CO22 assimilation leaves. The rateinset ( ain) and(a) demonstratesstomatalstomatal conductance the infection to water percentages. vapor ( bDuring) forfor J.J. the regiaregia measurements, controlcontrol (non-infected)(non- infected)the environmental andand A.A. erineaerinea conditions-infected-infected other leaves.leaves. than The light inset were in as (a) indemonstrates Figure 2. The thethe rectangular infectioninfection percentages. percentages.hyperbola of During During Smith the fittedthe measurements, measurements, the light response the the environmental environmental curve (a). conditions conditions other other than than light light were were as inas Figurein Figure2. The 2. The rectangular rectangular hyperbola hyperbola of Smith of Smith fitted fitted the the light light response response curve curve ( a). (a). Similar to the net assimilation rate, stomatal conductance increased with the increas- ing lightSimilar intensity toto thethe net(Figurenet assimilationassimilation 3b). The rate, rate,difference stomatal stomatal in conductance conductancestomatal conductance increased increased with betweenwith the the increasing increas-control andinglight infectedlig intensityht intensity leaves (Figure (Figurewas3b). particularly The3b). differenceThe differencelarge inat a stomatal lower in stomatal light conductance inte conductancensity of between 0– 400between μmol control controlm−2 and s−1 −2 −1 thanandinfected infectedat higher leaves leaves light was intensitywas particularly particularly of 40 large0– 1800large at aμmol at lower a lower m−2 light s −1light (Figure intensity intensity 3b). of Theof 0–400 0 –ratio400µmol μmolof stomatal m m−2s s−1 −2 −1 conductancethan atat higherhigher at lightlightlight intensity intensityintensities ofof of 400–1800 40 4000–1800 μmolµ μmolmol m−2 m ms−1−2 and ss−1 (Figure1800(Figure μmol 33b).b). m TheThe−2 s ratio−1ratio was ofof lower stomatalstomatal for −2 −1 −2 −1 controlconductance (15.13 at±at 0.15) lightlight than intensities intensities for infected of of 400 400 µleaves molμmolm (2.79 m−2s ±s −10.35 andand for 18001800 leavesµ μmolmol with m m−2 5.1% ss−1 was wasinfection, lower p for < 0.05).control (15.13 (15.13 ±± 0.15)0.15) than than for for infected infected leaves leaves (2.79 (2.79 ± 0.35± 0.35 for forleaves leaves with with 5.1% 5.1% infection, infection, p < 0.05).p < 0.05). 3.3. Responses of Foliage Chlorophylls and β-Carotene Contents to A. erinea Infection 3.3. Responses of Foliage Chlorophylls and β-Carotene Contents to A. erinea Infection 3.3. ResponsesBoth chlorophyll of Foliage a Chlorophyllsand b contents and reduced β-Carotene intensely Contents with to A. the erinea area Infectioninfected by the A. Both chlorophyll a and b contents reduced intensely with the area infected by the erineaBoth, e.g., chlorophyllthe average achlorophyll and b contents content reduced decreased intensely from with 742 the 4 mg area m infected−2 in non -byinfected the A. A. erinea, e.g., the average chlorophyll content decreased from 742 ± 4 mg m−2 in non- leaveserinea, toe.g., 554 the mg average m−2 in thechlorophyll leaves with content a considerable decreased frominfestation 742  4(Figure mg m−2 4a). in non The- infectedchloro- infected leaves to 554 mg m−2 in the leaves with a considerable infestation (Figure4a). The phyllleaves a /tob ratio 554 mgvaried m−2 betweenin the leaves 2.25 withand 2.75 a considerable (average ± SEinfestation = 2.49 ± 0.17)(Figure for 4a).all leaves The chloro- sam- chlorophyll a/b ratio varied between 2.25 and 2.75 (average ± SE = 2.49 ± 0.17) for all pledphyll and a/b didratio not varied depend between on the 2.25 degree and of2.75 infestation (average (±p SE > 0.05). = 2.49 ± 0.17) for all leaves sam- leaves sampled and did not depend on the degree of infestation (p > 0.05). pled and did not depend on the degree of infestation (p > 0.05).

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C 600 0 0 2 4 6 8 10 0 2 4 6 8 10 % leaf infected FigureFigure 4. 4. CorrelationsCorrelations of of chlorophyll chlorophyll aa ++ bb ((aa)) and and ββ--carotenecarotene ( (bb )) with with the the percentage percentage of of% A.A. l eerinea erineaaf in infectioninfectionfected in in J.J. regia regia leavesleaves (n = 25). The same leaves as used for physiological measurements (Figures 2, 5, and 6). The data have been fitted using (n = 25). The same leaves as used for physiological measurements (Figures2,5 and6). The data have been fitted using linear regression. linearFigure regression. 4. Correlations of chlorophyll a + b (a) and β-carotene (b) with the percentage of A. erinea infection in J. regia leaves (n = 25). The same leaves as used for physiological measurements (Figures 2, 5, and 6). The data have been fitted using linear regression.

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Forests 2021, 12, x FOR PEER REVIEW 7 of 12 β-Carotene concentration decreased from 34 mg m−2 in non-infected leaves to 25.69 mg m−2 in highly infected leaves (Figure 4b).

−2 3.4. Effectsβ-Carotene of A. erinea concentration Infection on decreased Stress-Elicited from Volatile 34 mg Emissions m in non -infected leaves to 25.69 mg m−2 in highly infected leaves (Figure 4b). The infection induced the emission of green leaves volatiles (GLV: (Z)-3-hexenol, (E)- 2-hexenal, (Z)-3-hexenyl acetate and 1-hexanol), monoterpenes (α-thujene, (E)-β-ocimene, 3.4. Effects of A. erinea Infection on Stress-Elicited Volatile Emissions 3-carene, 3-caren-2-ol) and a nerolidol-derived homoterpene 3-(E)-4,8-dimethyl-1,3,7- nonatrieneThe infection (DMNT). induced The emissions the emission of the of sum green of leavesgreen leaf volatiles volatiles (GLV: and ( Ztotal)-3- hexenol,monoter- (E)- 2penes-hexenal, increased (Z)-3- hexenylfrom nearby acetate zero and in control1-hexanol), leaves monoter to valuespenes as high (α-thujene, as ∼8.2 nmol(E)-β -mocimene,−2 s−1 3and-carene, 1.2 nmol 3-caren m−2 -s2−1- ol)(Figure and 5) a in nerolidol leaves with-derived a high homoterpeneinfected percentage. 3-(E) -4,8-dimethyl-1,3,7- Forests 2021, 12, 920 nonatriene (DMNT). The emissions of the sum of green leaf volatiles and total monoter-7 of 12 penes increased from nearby zero in control leaves to values as high as ∼8.2 nmol m−2 s−1 and 1.2 nmol10 m−2 s−1 (Figure 5) in leaves withGLV a high infected percentage. r = 0.971 Terpenes 8

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Figure 5. Correlations of monoterpene and green leaves volatiles emission rates with the percent- age of A. erinea infection in J. regia leaves (n = 25). Measurement conditions as in Figure 2. The data have been fitted using non-linear regression y = ax/(b + x). FigureFigure 5. 5.CorrelationsCorrelations of of monoterpene monoterpene and and green green leaves leaves volatiles volatiles emission emission rates rates with with the the percent- percentage ageof A.of InA. erinea addition,erineainfection infection the in emission J.in regia J. regialeaves of leaves characteristic (n =(n 25). = 25). Measurement Measurementstress terpenes, conditions conditions the monoterpene as inas Figurein Figure2. ( TheE 2.)-β The data-oci- data have havemene,been been fitted and fitted usingDMNT using non-linear also non scaled-linear regression positively regression y = with ay· x/(b= a thex/( +b x).infec + x).tion percentage (Figure 6).

In addition, the emission of characteristic stress terpenes, the monoterpene (E)-β-oci- mene, and DMNT also scaled positively with the infection percentage (Figure 6).

Figure 6. Correlations of the emission rates of the homoterpene 3-(E)-4,8-dimethyl -1,3,7-nonatriene (DMNT)FFigureigure 6. 6.and CorrelationsCorrelations the monoterpene of of the the emission emission (E)-β- ocimenerates rates of the of with thehomoterpene the homoterpene percentage 3-(E 3-() -of4,8E A.)-4,8-dimethyl-1,3,7-nonatriene-dimethyl erinea infection-1,3,7-nonatriene in J. regia leaves(DMNT)(DMNT) (n =and and25). the Env the monoterpeneironmental monoterpene (conditionsE) (-βE-)-ocimeneβ-ocimene during with with thethe measurementspercentage the percentage of A. as erinea of inA. Figure infection erinea 2.infection The in J. data regia in have J. regia beenleaves fitted (n =using 25). Environmentalnon-linear regression conditions y = a duringx/(b + x). the measurements as in Figure2. The data have been fitted using non-linear regression y = a·x/(b + x). 4. Discussion −2 4.1. Theβ -CaroteneInfluence of concentrationAceria erinea Infection decreased on Photosynthetic from 34 mg Characteristics m in non-infected of Juglans leavesregia to 25.69 mg m−2 in highly infected leaves (Figure4b).

3.4. Effects of A. erinea Infection on Stress-Elicited Volatile Emissions The infection induced the emission of green leaves volatiles (GLV: (Z)-3-hexenol, (E)- 2-hexenal, (Z)-3-hexenyl acetate and 1-hexanol), monoterpenes (α-thujene, (E)-β-ocimene, 3-carene, 3-caren-2-ol) and a nerolidol-derived homoterpene 3-(E)-4,8-dimethyl-1,3,7- nona- triene (DMNT). The emissions of the sum of green leaf volatiles and total monoterpenes increased from nearby zero in control leaves to values as high as ∼8.2 nmol m−2 s−1 and 1.2 nmol m−2 s−1 (Figure5) in leaves with a high infected percentage. In addition, the emission of characteristic stress terpenes, the monoterpene (E)-β- ocimene, and DMNT also scaled positively with the infection percentage (Figure6). Forests 2021, 12, 920 8 of 12

4. Discussion 4.1. The Influence of Aceria erinea Infection on Photosynthetic Characteristics of Juglans regia The general response of plants to infection by different pathogens includes reduc- ing leaf net assimilation rate [22,25,28,44]. According to previous studies, our data also demonstrated a substantial decrease in the net assimilation rate for infected plants. Notably, the relative reduction in net assimilation rate observed in A. erinea-infected J. regia leaves was much greater (up to 80%) than the degree of infection (up to 10%, Figure2a). This indicates that erineum-formation led to profound physiological modifications in both erineum-infected and non-infected leaf areas. Furthermore, it suggests that the impact of gall- and erineum-forming mites can be much more significant than generally thought based on a relatively small fraction of the area infected in individual leaves. The relative reduction in leaf net assimilation rate in erineum-infected leaves can be due to stomatal and non-stomatal (reductions in leaf photosynthetic capacity) factors. In the present study, the relative decrease in net assimilation rate was related to reduced stomatal conductance (Figure2b) and leaf water loss. An analogous negative influence of gall- and erineum-forming mites on water loss and photosynthesis has been found in Vitis vinifera cultivars infected with erineum-forming mite Colomerus vitis [45], Acer saccharum trees galled by the mite Vasates aceriscrumena [46], in Alnus glutinosa trees infected by , and Tilia cordata trees infected by E. exilis [20]. The relative reduction in stomatal conductance in pest-infected leaves could be explained by impaired stomatal function in gall- and erineum-covered leaf regions and surrounding leaf tissues due to pathogenic infection or formation of necrotic areas [17,28,47]. However, stomatal conductance is typically reduced less than the net assimilation rate, resulting in reduced leaf water use efficiency [17,48], as observed in our study. The evidence of a greater reduction in net assimilation rate than in stomatal con- ductance suggests that the infection also reduced leaf photosynthetic capacity. Typically, leaf photosynthetic capacity is significantly reduced in the infected leaf regions, reflecting altered leaf anatomy and reduced photosynthetic pigment content [46], as observed in our study (Figure4). A significant reduction in leaf chlorophyll content has been shown in the horn-shaped galls induced by a cecidomyiid on Copaifera langsdorffii leaves [49] and leaves of Cinnamomum tamala with Aceria doctersi galls [50]. The decrease in chlorophyll contents has been connected with a dilution of pigments by cell hypertrophy in leaves with galls [51,52]. However, in our study, the relative decrease in pigment content in J. regia at given infection severity was much less than in the net assimilation rate (cf. Figures2 and4). In fact, there is evidence that the mites might also affect the sink–source relationships (by interaction with others sinks within integrated physiological units) during leaf growth and thereby alter photosynthetic biomass accumulation per unit leaf area and whole leaf photosynthesis rate [53]. Such a sink–source impact has been demonstrated in Populus × petrovskiana infected by petiole gall aphids; the aphid galls are formed on petioles, and thus, the aphids do not directly affect leaf lamina, but nevertheless, leaf photosynthesis rate was strongly reduced in leaves with infested petioles (Ye et al. 2019). It is plausible that the disproportionately more significant reduction in net assimilation rate at the given degree of infection in J. regia is also associated with altered leaf development and decline of photosynthetically active biomass per unit area in infected leaves.

4.2. Emission of Volatile Organic Compounds from Leaves Infected with A. erinea The green leaf volatiles (GLV, C5 and C6 alcohols, aldehydes, and ketones) are spe- cific elicitor molecules emitted from damaged plant membranes, usually after herbivory, pathogen, or oxidative stresses [28,29,54–56]. In the case of A. erinea infection in J. regia, GLV emissions is amplified by increasing the degree of infection (Figure4a). Similar scaling of GLV emissions with the degree of infection of gall- or erineum- forming arthropods has also been observed in other studies [17,20,25]. However, quantitatively, the rate of GLV emission observed in these studies was less, except in Quercus robur leaves infected by Forests 2021, 12, 920 9 of 12

Neuroterus anthracinus wasp galls where the rates of GLV emission were similar to J. regia leaves with A. erinea infection of 1–2% [17] (Figure5). A sustained GLV emission could be a direct response to the immediate sites of im- pact during mite feeding, reflecting the free fatty acids discharge from damaged plant membranes and activation of at the sites of feeding within the erinea [57,58]. However, once formed, the primary GLV can translocate to other leaf regions with the tran- spiration stream and can be derivatized before the release [59]. In the case of infection with A. erinea, GLV emissions were dominated by 1-hexanol, suggesting that derivatization of a part of primary aldehydes could have occurred in non-damaged leaf parts. Analogously, a large share of derivatized GLV was found in Quercus robur leaves infected with and Cynips spp. [17] and in Alnus glutinosa infected by Eriophyes inangulis mites and Tilia cordata infected by E. tiliae mites [20]. The leaves of J. regia do not emit monoterpenes in non-stressed conditions, but this species has glandular trichomes on the leaf surface, and leaf essential oil extractions contain different mono- and sesquiterpenes [60,61]. Thus, a certain fraction of monoterpene release in A. erinea-infected mites might be originated from terpene released upon breakage of glandular trichomes. However, given that the erinea harboring the mites are primarily covered by non-glandular hypertrophic trichomes, the contribution of glandular trichomes to terpene release in mite-infected leaves is likely minor. On the other hand, de novo syn- thesis of monoterpenes is typically elicited in response to pest and parasite attacks as a part of the induced suite of traits involved in direct and indirect defense (for review, see [62,63]). As a part of the direct defense, terpenes can be toxic to infecting organisms, including mites [63,64]. Volatile terpenes also play a compelling role in indirect defense by attracting predators of plant-attacking insects and mites [63,65,66]. In the case of J. regia leaves, there is only limited information on arthropod-induced terpene blends. Infestation of J. regia leaves by Meliboeus ohbayashii ssp. primoriensis larvae induced emissions of monoterpenes limonene, sabinene, β-phellandrene, α-pinene, and β-pinene and sesquiterpenes germa- crene D, (E,E)-α-farnesene [67]. In our study, J. regia leaves infected with A. erinea emitted monoterpenes α-thujene, (E)-β-ocimene, and 3-carene and its oxidized derivative 3-caren- 2-ol, and total monoterpene emissions scaled with the percentage of the leaf area infected (Figure4b). In addition, the emissions of two characteristic terpenes de novo synthesized in stressed leaves, (E)-β-ocimene and the homoterpene DMNT, which also scaled positively with the degree of leaf area damage (Figure6), suggesting that the positive scaling of total terpene emission with the degree of infection in our study (Figure4b) primarily reflected de novo synthesis of terpenes. The significant differences in the induced terpene emission blends from J. regia infected by the mite A. erinea in our study and the buprestid insect M. ohbayashii ssp. primoriensis [67] suggest that the induced terpene composition provides informative insights for identifying the infecting organism. We suggest that further work with different walnut-infecting organisms is needed to characterize the modification of the emission blend and the degree of elicitation of volatile emissions as determined by various leaf-attacking arthropods.

5. Conclusions Our study demonstrates the infestation of walnut leaves with Aceria erinea strongly affects the plant’s metabolic and physiological processes. In addition to visual symptoms, the infection led to a decline in photosynthetic characteristics, emission of volatile organic compounds, and a decrease in chlorophylls and carotene contents. All chemical and physi- ological modifications were quantitatively related to the percentage of the leaf area infected. In contrast, the decline in photosynthetic characteristics was much more significant than could be predicted based directly on the infected leaf area. We conclude that A. erinea infection leads to systemic alterations in J. regia leaf physiological activity.

Author Contributions: Conceptualization, L.C., Ü.N., and D.M.C.; methodology, L.C.; validation, L.C., Ü.N., and D.M.C.; formal analysis, C.P., A.L., L.C., S.B.; investigation, C.P., A.L., S.B., L.C.; resources, L.C.; data curation, C.P., A.L., S.B.; writing—original draft preparation, C.P., A.L., S.B., Forests 2021, 12, 920 10 of 12

L.C., Ü.N., and D.M.C.; writing—review and editing, Ü.N., L.C., and D.M.C.; visualization, L.C.; supervision, L.C., D.M.C.; project administration, L.C., D.M.C.; funding acquisition, L.C., D.M.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by CNFIS-UEFISCDI, project number PN-III-P4-ID-PCE-2020- 0410. Conflicts of Interest: The authors declare no conflict of interest.

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