Leaf gas exchange and chlorophyll a fluorescence in Hevea brasiliensis in response to Pseudocercospora ulei infection

Accepted Manuscript Leaf gas exchange and chlorophyll a fluorescence in Hevea brasiliensis in response to Pseudocercospora ulei infection Armando Sterling, Luz Marina Melgarejo PII:

S0885-5765(18)30178-4

DOI:

10.1016/j.pmpp.2018.07.006

Reference:

YPMPP 1349

To appear in:

Physiological and Molecular Plant Pathology

Received Date: 4 June 2018 Revised Date:

19 July 2018

Accepted Date: 23 July 2018

Please cite this article as: Sterling A, Melgarejo LM, Leaf gas exchange and chlorophyll a fluorescence in Hevea brasiliensis in response to Pseudocercospora ulei infection, Physiological and Molecular Plant Pathology (2018), doi: 10.1016/j.pmpp.2018.07.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title Page

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Leaf gas exchange and chlorophyll a fluorescence in Hevea brasiliensis in response to Pseudocercospora ulei infection

Armando Sterling1*, Luz Marina Melgarejo2

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Laboratorio de Fitopatología, Instituto Amazónico de Investigaciones Científicas Sinchi, Florencia – Colombia. 2

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Laboratorio de Fisiología y Bioquímica Vegetal, Departamento de Biología, Universidad Nacional de Colombia - Sede Bogotá.

*Corresponding author: [email protected], [email protected]

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Conflict of Interest: The authors certify that no conflict of interest exists.

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South American leaf blight (SALB), the main disease found in the rubber tree crops of Latin America, is caused by the fungus Pseudocercospora ulei. This study aims to determine the photosynthetic response of two clones of H. brasiliensis with different resistances to SALB from P. ulei under controlled conditions, by means of a temporal analysis of gas exchange and chlorophyll a fluorescence. The results show that the effect on photosynthesis was proportional to the temporal progress and intensity of the disease symptoms to this effect, the maximum significant decrease (p <0.05) in photosynthetic rates in the clone FX 3864 (susceptible) (88.3%) and in FX 4098 (moderately resistant) (45.2%) was observed 8 days after inoculation in B leaflets that were 18 days old. Meanwhile, a significant difference was found between the two clones’ ability to capture, use, and dissipate light energy through photosystem II, which was evidenced by the minimum photosynthetic activity registered in the susceptible clone.

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Keywords: rubber tree clones; South American Leaf Blight; photosynthesis; controlled conditions

Introduction

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The South American leaf blight (SALB), spread by the fungus Microcyclus ulei (P. Henn.) V. Arx and recently renamed Pseudocercospora ulei, is the main disease affecting rubber crops in Latin America [1]. This disease is of great importance because it produces repeated premature defoliation, decreases the production of latex by 20% to 75%, and can lead the plant to die [2,3]. According to Gasparotto et al. [4], the first phase of the disease – the infectious phase – occurs after 4 to 10 days, depending on the rubber tree’s clone. Dusty conidial damage inflicted by P. ulei appears in the young foliar stages B (10 -18 days old) and C (19 to 30 days old)[5]. In the second phase (two months after infection), D Leaflets with physiological maturity (60-90 days old) exhibit asexual stromal damage (pycnidia). Finally, in the third phase (three to four month after infection), sexual stromal damage appears in D Leaflets approaching senescence (90 to 120 days old), which carry ascospores with which a new disease cycle will begin. The use of clones resistant to SALB is currently the main strategy for disease management [6].

Studies in SALB have primarily concentrated on biochemical and molecular mechanisms related to disease resistance [3,6], the type of clonal reaction [7–9], and the impact of environmental conditions on the epidemiological dynamics of the disease. Consequently, many of the physiological aspects involved in the interaction H. brasiliensis –P. ulei are still unknown. Because P. ulei is a hemibiotrophic pathogen that mainly colonizes living foliar tissue, it is assumed that from the time the infectious process begins, major changes must occur over time in the physiology of rubber resulting from host defense mechanisms [3] and the variation of symptoms and signs produced by the pathogen over time [10]. In other hemibiotrophic hostpathogen interactions, major physiological effects on the host have mainly been observed in photosynthesis [11,12].

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Abstract

Photosynthetic parameters related to gas exchange and chlorophyll a fluorescence are considered noninvasive indicators of photosynthetic response under conditions of pathogen-induced biotic stress [13–15]. To this effect, the analysis of these indicators has shown that infection from pathogens cause reductions in photosynthetic rates as well as modifications in the photosynthetic apparatus [15–18]. These changes may be due to the negative regulation or damage of the photosynthetic apparatus [15,19]. For example, analysis of chlorophyll a fluorescence has shown that the photochemical efficiency of photosystem II (PSII) is reduced by infection [15,16,20].

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Given that the use of clones resistant to SALB constitutes one of the most promising strategies for disease management [6,21,22], it is assumed that the physiological effects of P. ulei on the H. brasiliensis must be less evident in materials that are less susceptible to the disease. Considering that the use of genetic material with some level of resistance to foliar pathogens could reduce negative effects on photosynthesis, this study considered the following hypotheses: (a) the photosynthetic performance of H. brasiliensis clone FX 4098 in response to P. ulei infection is superior to that observed in

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the clone FX 3864; and (b) under controlled conditions of infection and regardless of the clone used, changes in photosynthesis are proportional to the amount of time the disease has progressed. To prove these hypotheses, this study aimed to use analysis of gas exchange parameters and chlorophyll a fluorescence to determine the photosynthetic response of two clones of H. brasiliensis (FX 4098 and FX 3864) with different resistance to SALB infected by P. ulei under conditions of controlled inoculation.

Materials and methods

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2.1. Adaptation of experimental conditions

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The experiment was conducted in the temperature-controlled room of the Sinchi Phytopathology Laboratory of the Amazonian Institute of Scienctific Research in Florencia, Caqueta (Colombia), located at coordinates 1° 37' 03" N and 75° 37' 03" W [23]. 10 cubicles (inoculation booths) were set up inside the temperaturecontrolled room. The air temperature was set to 23 °C and relative humidity to 90 - 95%, and a photoperiod of 12/12 h was established (PAR = 340 µmol photons m-2 s-1) [7,9]. 2.2. Vegetal material

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FX 4098 clones that were highly productive, vigorous and moderately resistant to P. ulei were used [9,21], as well as clones of FX 3864 that were susceptible to P. ulei [7]. For each clone of H. brasiliensis, four 4-monthold seedlings (two leaf stories) were grown in bags with a capacity of 7 kg in soil from the region of Caqueta. 2.3. Inoculum source and controlled inoculation

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CVI2 isolation of P. ulei, belonging to the isolation bank of the Phytopathology Laboratory of the Sinchi Institute, was used [22]. The isolate was maintained in growth medium (MC) – adapted from Mattos et al. [7] – kept in the dark at 24 °C, and sporulated in M4 medium (potato dextrose agar - PDA) under a photoperiod of 12/12 h. A suspension of inoculum measuring 2 x 105 conidia mL-1 was prepared in sterile distilled water and sprayed on the underside of four 10-day-old leaves for each of the five plants of each clone, using an aerograph airbrush adjusted to an electric compressor calibrated at 4.5 Pa pressure [9]. After inoculation, the plants were kept in darkness for 24 hours and thereafter were subjected to a photoperiod of 12/12 h until day 20 (PAR= 11.5 µmol photons m-2 s-1), at a temperature 23 °C and a relative humidity between 90 and 95%. 2.4. Disease severity assessment

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The severity of SALB (P. ulei) was evaluated at 0, 4, 8, 12, 16, and 20 days after infection (DAI) using the scale adapted by Gasparotto et al. [4], defined as the percentage of a foliar area, and valued on a scale from 0 to 4: 0 = null, (0% foliar area affected); 1 = low (0.2 to 5% foliar area affected); 2 = medium (6 to 15% foliar area affected); 3 = high (18 to 30% of foliar area affected); 4 = very high (40 to 100% foliar area affected). Likewise, the area under the disease progress curve (AUDPC) of SALB (through the trapezoidal integration of the severity progress curve by way of the formula proposed by Campbell and Madden [24].

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2.5. Gas exchange measurements Net CO2 assimilation rate (A) (µmol CO2 m-2 s-1), transpiration rate (E) (mmol H2O m-2 s-1), and stomatal conductance (gs) (mol H2O m-2 s-1) were measured using a portable equipment for the measurement of IRGA gas exchange, which uses an open system of measurement (TPS 2 PP Systems, USA). Water use efficiency extrinsic was also calculated (WUEe) expressed as WUEe = A/E, which determines the balance between water loss and CO2 uptake [25]. The equipment was programmed under laboratory conditions at a controlled temperature (23 °C), a saturating relative humidity (90 - 95%), CO2 concentrations (400 ppm), and a saturating (1547 µmol photons m-2 s-1) photosynthetic photon flux density (PPFD). The data of saturating PAR was determined according to previously conducted light curves. For each clone, evaluations were conducted between 9:00 and 11:00 h on two trifoliate leaves (in the central leatlet of each leaf) of the second leaf story in each the five plants used in each treatment (Inoculated [I] and Non-Inoculated [NI]). Each leaf

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was labeled so as to be able to take repeated measurements over time at 0, 4, 8, 12, 16, and 20 DAI to cover the progress of the infectious phase of the disease.

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2.6. Chlorophyll a fluorescence measurements

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Chlorophyll a fluorescence parameters were estimated immediately after gas exchange measurements were taken using a pulse modulated portable fluorometer (Hansatech, Hoddesdon, England). The evaluations were conducted on the same leaflets that were used to measure gas exchange. Prior to the recording of fluorescence parameters, the measuring point of each leaflet was acclimated to the dark for 30 min. Once this procedure was completed, the vegetal tissue was exposed to a weak beam of modulated light (0.03 µmol m-2 s-1) in order to determine initial fluorescence (F0). Then, a pulse of saturating white light 6000 µmol m-2s-1 was applied for 1 s to ensure maximum fluorescence emission (Fm), from which the Fv/Fm = (Fm – F0)/Fm parameter was calculated (maximum quantum yield of photosystem II, PSII). In addition, the following parameters were calculated under actinic light (1000 µmol m-2.s-1): efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm'), photochemical quenching coefficients (qP), non-photochemical quenching coefficients (NPQ), and electron transport rate (ETR). The ETR was calculated using the equation: ETR = ФPSII x PPFD x f x α [26].

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2.7. Experimental design and data analysis

The experiment consisted of a completely randomized design with a 2 x 2 factorial arrangement, where Factor A corresponded to the clone (moderately resistant clone - FX 4098 and susceptible clone - FX 3864), Factor B corresponded to the treatment (inoculated - I and non-inoculated - NI), and the experimental unit (repetition) was each of the five rubber plants per factorial combination. The experiment was realized twice.

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Results

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The following factors were adjusted to a general linear model: clone (FX 3864 and FX 4098), treatment (I and NI), sampling time (0, 4, 8, 12, 16, and 20 DAI), and their interactions, with the aim of analyzing the following responses variables: severity, A, E, gs, WUEe, Fv/Fm, F'v/F'm, qP, NPQ, and ETR. The residual variance was modeled in such a way so as to allow the consideration of different variances (Heteroscedasticity, H) per sampling time, while the residual correlation for subsequent observations made on the same plant was considered using compound symmetry (CS), first-order autoregressive (AR1), and unstructured (UN) models. The Akaike information criterion (AIC), Bayesian information criterion (BIC), and the LogLik function were used to select the structure of residual variances and correlations [27]. The adjustment was made using the lme function of the nlme library [28] of R package version 3.4.0 [29] on the InfoStat interface [27]. The analysis of fixed factors and their interactions for the comparison of means test was done using Fisher’s LSD test for multiple comparisons (α = 0.05). The correlation coefficients (Pearson’s test) between different variables were measured the susceptible clone’s (FX 3864) inoculated plants at foliar stages B (10 to 18 days old) and C (19 to 30 days old), due to the fact that this material exhibited the highest levels of disease severity.

3.1. SALB severity and AUDPC Significant differences were found in the severity of SALB and in the area under the disease progress curve (AUDPC) (both variables, p <0.05) in the two rubber tree clones (Table 1). The severity of SALB in clone FX 3864 was significantly higher over time relative to that observed in FX 4098, where the difference was greater beginning at 12 DAI (Fig. 1A). The AUDPC in clone FX 3864 was 42.8% greater than in clone FX 4098 (p <0.05) (Fig. 1B). The non-inoculated (NI) plants for both clones showed no evidence of infection and were consequently used as control plants.

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Table 1 Analysis of variance of the effects of clone (C), treatment (T), sampling time (TM), and their interactions, on severity, area under the disease progress curve (AUDPC), net CO2 assimilation rate (A), stomatal conductance to water vapor (gs), transpiration rate (E), water use efficiency extrinsic (WUEe), maximum quantum yield of photosystem II (PSII) (Fv/Fm), efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm'), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and electron transport rate (ETR). F based P values TM

CxT

C x TM

<0.001

-

0.031

<0.001

T -

<0.001

<0.001

<0.001 <0.001

<0.001 <0.001

<0.001 <0.001

<0.001 <0.001

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Variablesa C

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Severity

<0.001

AUDPC A gs

<0.001

<0.001

<0.001

<0.001

WUEe Fv/Fm

<0.001 0.021

<0.001 <0.001

<0.001 0.001

<0.001 0.008

Fv'/Fm'

0.535

0.004

<0.001

0.601

qP NPQ

0.003 0.730

0.191 0.466

<0.001 0.053

0.874 0.616

C x T x TM

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<0.001 <0.001

<0.001 <0.001

<0.001

<0.001

<0.001

<0.001 0.419

<0.001 <0.001

<0.001 0.005

0.003

0.004

0.024

<0.001 0.179

0.031 0.135

0.007 0.317

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E

<0.001 <0.001

T x TM

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0.001 0.115 <0.001 0.946 <0.001 0.014 0.470 ETR For the variables of the disease, the best model was the compound symmetry model with homogeneity of variance (CS); for the physiological variables, the model selected was the compound symmetry with heterogeneity of variance (CSH). a

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Fig. 1. SALB severity progress curve in plants inoculated with Pseudocercospora ulei. (A) and area under the disease progress curve (AUDPC) (B), for two clones of the rubber tree (Hevea brasiliensis) under controlled inoculation. DAI ≤ 4 ≤ 8 B Leaflets (14 to 18 days old). 8
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3.2. Photosynthetic parameters

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Significant differences were found in A, gs, E, and WUEe between the two rubber tree clones, between treatments (I and NI), and over time (DAI). Additionally, all interactions between these factors were significant (Table 1). For clone FX 4098 (moderately resistant), significant differences were found in A between inoculated plants (I) and non-inoculated plants (NI) at 8 DAI (Fig. 2A), and at 8, 16, and 20 DAI for clone FX 3864 (susceptible) (Fig. 2B). In clone FX 4098 the major effect on A occurred during foliar stage B at 8 DAI in inoculated plants, with a decrease of 45.2% as compared to non-inoculated plants. This was different to what was observed during foliar stage C (≥ 12 DAI) where there was no significant decrease in A. For clone FX 3864, the decrease of A was 88.3% at 8 DAI (stage B) and 70.6% at 20 DAI (stage C) in inoculated and non-inoculated plants, respectively.

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Similar to what was recorded for A, the greatest decreases in WUEe occurred at 8 DAI in both clones (71.1% in FX 4098 and 93.5% in FX 3864) (Fig. 2G and 2H, respectively). For inoculated plants of clone FX 3864, the clone most greatly affected by SALB (< A), the gs and E parameters were significantly lower – 74.6% at 16 DAI and 58.8% at 20 DAI, respectively – than in non-inoculated plants (Fig. 2D, F).

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Significant differences were observed between the FX 4098 and FX 3864 clones from 4 to 20 DAI for A and for gs (except at 8 DAI) (Fig. 2A-D), and at 12 and 16 DAI for E (Fig. 2E, F). Between 12 and 16 DAI, significant difference was observed in the mean values of WUEe in the two rubber tree clones (Fig. 2G, H).

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For the moderately resistant clone (FX 4098), it was found that treatment (I and NI) had a significant effect on the Fv/Fm, Fv'/Fm' and qP parameters at 20 DAI, and on qP at 16 DAI (in all cases, during foliar stage C) (Fig. 3A, C, E). In this instance, the average values of Fv/Fm, Fv'/Fm', and qP decreased significantly by 5.8%, 5.2%, and 20.8% respectively in inoculated plants as compared to non-inoculated plants (20 DAI). There was also a 30.7% decrease at 16 DAI in the mean of qP in inoculated plants as compared to non-inoculated plants at. There were no significant differences in the parameters NPQ and ETR between inoculated and non-inoculated (Fig. 3G, I). Significant differences were found in Fv/Fm, Fv'/Fm', qP, and ETR in at least one of the factors (clone, treatment, and DAI), as well as in some of the interactions among factors (Table 1). Neither these factors nor their interactions had a significant effect on the mean value of NPQ.

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For the susceptible clone (FX 3864), the average values of Fv/Fm decreased significantly by 9.5%, 9.6%, and 11.9% in inoculated plants as compared to non-inoculated plants at 8, 16, and 20 DAI, respectively (Fig. 3B). In addition, Fv'/Fm' decreased significantly by 12.2% and 6.7% in inoculated plants as compared to noninoculated plants at 8 and 20 DAI, respectively (Fig. 3D). Treatment (I and NI) did not have a significant effect on the qP, NPQ, and ETR parameters (Fig. 3F, H, J). No significant differences were found in Fv/Fm between the FX 4098 and FX 3864 clones (Fig. 3A, B). There were significant differences in average Fv'/Fm' between clones at 12 DAI, with higher values for FX 4098 (Fig. 3C, D). Differences in qP and ETR between clones occurred at 0, 4, and 20 DAI (Fig. 3E, F, I, J). Additionally, significant differences occurred in qP at 16 DAI.

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Fig 2. Net CO2 assimilation rate (A) (A, B); stomatal conductance to water vapor (gs) (C, D); transpiration rate (E) (E, F); and water use efficiency extrinsic (WUEe) (G, H) for leaflets of the rubber plant (Hevea brasiliensis) clones FX 4098 (moderately resistant) (A, C, E, G) and FX 3864 (susceptible) (B, D, F, H), inoculated (I) and non-inoculated (NI) with Pseudocercospora ulei under controlled conditions. 4 ≤ DAI ≤ 8, B Leaflets (14 to 18 days old). 8
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Fig 3. Maximum quantum yield of photosystem II (PSII) (Fv/Fm) (A, B); efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm') (C, D); photochemical quenching coefficient (qp) (E, F); nonphotochemical quenching coefficient (NPQ) (G, H); electron transport rate (ETR) (I, J) for leaflets of rubber plants (Hevea brasiliensis) of the clones FX 4098 (partially resistant) (A, C, E, G, I) and FX 3864 (susceptible) (B, D, F, H, J), inoculated (I), and non-inoculated (NI) with Pseudocercospora ulei under controlled conditions. 4 ≤ DAI ≤ 8, B Leaflets (14 to 18 days old). 8
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3.3. Pearson’s correlation Pearson’s correlation analysis conducted on inoculated plants of the susceptible clone (FX 3864) (the genetic material that exhibited the highest levels of severity of SALB and the greatest physiological alteration) showed a positive correlation of A with the parameters gs, E, and WUEe in both foliar stages (Table 2). In both phenological stages, there was also a negative correlation of SALB severity with all gas exchange parameters; for these, the only correlation that was not significant was between severity and WUEe in foliar stage C.

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Table 2. Pearson’s correlation coefficients for foliar stage B (10 to 18 days old) (above the diagonal) and foliar stage C (19 to 30 days old) (below the diagonal), between SALB severity, A, gs, E, and WUEe as measured in rubber tree plants (Hevea brasiliensis) of the clone FX 3864 (susceptible) inoculated with Pseudocercospora ulei gs

Severity -0.76** 0.94** -0.63** 0.95** 0.86** -0.87** E 0.87** 0.85** -0.80** WUEe … -0.54* -0.58* -0.58* -0.38ns Severity A, Net CO2 assimilation rate; gs, stomatal conductance to water vapor; E, transpiration rate; water use efficiency extrinsic, WUEe 0.60** …

E 0.86** 0.73** … 0.70**

WUEe 0.98** 0.62** 0.92** …

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Parameter A gs

* P <0.05; significant; ** P <0.01, very significant; ns, not significant

Regarding chlorophyll a fluorescence parameters, significant positive correlations were observed of Fv/Fm with qP, NPQ, and ETR during foliar stage B (10 to 18 days), while during foliar stage C (19 to 30 days), Fv/Fm only showed significant positive correlatiosn with Fv'/Fm' and ETR (Table 3). Fv/Fm was the only parameter that was negatively correlated with severity in both foliar stages.

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Table 3. Pearson’s correlation coefficients for foliar stage B (10 to 18 days old) (above the diagonal) and foliar stage C (19 to 30 days old) (below the diagonal), including SALB severity, Fv/Fm, Fv'/Fm', qP, NPQ, and ETR as measured in rubber tree plants (Hevea brasiliensis) of the clone FX 3864 (susceptible) inoculated with Pseudocercospora ulei

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Parameters NPQ ETR Severity Fv/Fm Fv'/Fm' qP … 0.87** 0.48ns 0.11ns 0.51* -0.57* Fv/Fm … 0.35ns 0.57* 0.34ns 0.60* -0.61* Fv'/Fm' … 0.50* 0.11ns 0.52* 0.99** -0.09ns qP … 0.59* 0.26ns 0.11ns 0.53* -0.01ns NPQ … 0.53* 0.24ns 0.96** 0.20ns -0.12ns ETR … -0.58* -0.38ns -0.34ns -0.57* -0.37ns Severity Fv/Fm, maximum quantum yield of photosystem II (PSII); Fv'/Fm', efficiency of excitation energy capture by open PSII reaction centers; qP and NPQ, photochemical and non-photochemical quenching coefficients, respectively; and ETR, electron transport rate. * P <0.05; significant; ** P <0.01, very significant; ns, not significant

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4.

Discussion

The results show that the parameters for gas exchange and chlorophyll a fluorescence in the moderately resistant clone (FX 4098) were not very affected after inoculation with P. ulei. This behavior is associated with the good agronomic performance that this material has already exhibited under field conditions [21]. In

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contrast, in the susceptible clone (FX 3864), gas exchange parameters were strongly affected by the infection of P. ulei; this is similar to the effects reported in other species [16].

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In this study, the infection of P. ulei in young rubber tree leaflets [(stages B (10 to 18 days old) and C (19 to 30 days old)], had significant negative effects on photosynthesis A in plants susceptible to the pathogen. As SALB severity increased over time, the values of A decreased significantly, but this effect was less evident in clones with reduced susceptibility to SALB (FX 4098). This favorable behavior in A in the clone FX 4098 could be explained by the lesser symptoms exhibited over time by this material, which has been demonstrated by Sterling and Melgarejo [10], who found a significant decrease in the temporal progression of symptoms and signs in clone FX 4098 as compared to that observed in FX 3864 from 4 DAI. The decrease in A caused by the heightened severity of foliar diseases has been demonstrated in other species [11,12,15,17,18].

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Low photosynthesis rates in immature foliar stages (B and C) may be associated with processes such as stomatal resistance, elevated respiration [30] and high points of CO2 compensation. However, other features such as the accumulation of dry matter, chlorophyll content, and stomatal conductance [31] increases with the age of the leaves, reaching peak values in mature leaflets. In this study, the imbalance of these parameters could be related to the decrease in net photosynthesis in young leaflets of the two clones evaluated.

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In the pathosystem H. brasiliensis - P. ulei, during the more advanced stages of the disease (8 DAI, B Leaflets, and 16 - 20 DAI, C Leaflets), a significant decrease (p <0.01) in gs occurred, while there was a significant decrease (p <0.01) in A in the susceptible clone (FX 3864). Pinkard and Mohamed [11] and Alves et al. [15] described a similar effect in eucalyptus pathosystems (Eucalyptus globulus) - Mycosphaerella sp. and E. urophylla - Puccinia psidii under greenhouse conditions. According to Bacelar et al. [32] stomatal closure due to reduced availability of water in the diseased tissue compromises the electron transport chain (ETR) and reduces CO2 fixation in the Calvin cycle.

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This suggests that a decrease in A is caused by the activity of the disease, which probably leads to biochemical limitations in the assimilation and fixation of CO2 in chloroplast stroma during the Calvin cycle [16,33]. These reductions in A are probably due to the low activity of photosynthetic enzymes such as Rubisco [33], or enzymes involved in the degradation of photoasimilates. This would explain the higher values of A observed in this study for the inoculated plants of clone FX 4098 as compared to the FX 3864. In most diseases, A suffers a decrease from the beginning of the infection [34]. However, in this study photosynthesis was affected at 8 DAI in leaflets with symptoms of SALB at levels of severity '2' and '3' in the clones FX 4098 and FX 3864, respectively. That is, in addition to being proportional to the progress of SALB severity over time, the decrease in A was impacted by the level of resistance of the rubber tree clone.

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The negative values of net photosynthesis in B Leaflets reported in this study may be related to low chlorophyll contents, low Rubisco activity, or because the leaflets had not yet reached physiological maturity [3,31]. On the other hand, Miguel et al. [31] found high photosynthetic rates and considerable stomatal conductance in fully developed leaves in clones RRIM 600, PB 235, and GT 1 (D Leaflets 52 to 57 days old). In contrast, both studies reported negative rates of net photosynthesis and low stomatal conductance low in Bstage leaflets. Based on these statements, it is possible that in this investigation, the stomata did not fully develop during foliar stage B, implying a low stomatal conductance and therefore the absence of a positive net photosynthesis.

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Decreases in transpiration (E) in inoculated plants of the susceptible clone could be related to decreases in gs values and therefore could be associated with stomatal closure. Several studies have demonstrated similar decreases in E and gs in pathosystems E. urophylla - P. psidii [15], sorghum (sorghum bicolor) Colletotrichum sublineolum, [35] bean (Phaseolus vulgaris) - C. lindemuthianum [12], and wheat (T. aestivum) - P. oryzae [17]. E decreases in susceptible rubber tree plants could also be related to the increase in the intensity symptoms – especially necrotic symptoms – beginning at 8 DAI, due to the massive colonization of vegetal tissue by P. ulei. This idea is supported by Resende et al. [35] and Rios et al. [17], who found an association between the

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decrease in E and the symptoms of desiccation and wilting observed in sorghum and wheat leaves highly colonized by C. sublineolum and by P. oryzae, respectively.

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Also, decreases in water use efficiency extrinsic (WUEe) were associated with decreases in A, E, and gs in both foliar stages. These decreases in WUEe therefore imply an imbalance between CO2 uptake and water loss through transpiration [32,36]; that is, an adaptation in the strategies used by susceptible rubber tree plants for water conservation in foliar tissues, caused by infection by P. ulei. For healthy leaflets of both rubber tree clones in this study, the highest photosynthetic rates and the most efficient use of extrinsic water occurred in C Leaflets (19 to 30 days old). This coincides with the findings of Miguel et al. [31], who observed significant increases in stomatal conductance, chlorophyll content, carboxylation efficiency, and WUE in C leaflets 47 days in age. Vinod et al. [37] also reported significant increases in chlorophyll content a, b, total, and a/b ratio during the process of foliar ontogeny in five rubber tree clones under stress induced by low temperatures.

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The lowest observed negative impact on gas exchange parameters in the clone FX 4098 could be explained by more than the lower susceptibility to attack by P. ulei (least severe); it is likely that an increase in the activity of some antioxidant enzymes within active biochemical defense mechanisms in the plant in response to infection limits cell damage throughout the process of infection [35]. In the susceptible clone (FX 3864), infection by P. ulei caused a slight impairment in the maximum quantum yield of photosystem II (PSII) (Fv/Fm), decreasing the mean from 0.83 to 0.75, suggesting a possible photoinhibition in the PSII reaction center [13,38–40]. There was also a simultaneous decrease in the efficiency excitation energy capture by open PSII reaction centers (Fv'/Fm'), as well as a decrease in the photochemical dissipation of absorbed light (qP) (8 DAI). This indicates that the inoculated plants significantly reduced the clone’s ability to capture and use light energy. Similar photochemical dysfunction has been reported for other pathosystems, such as in E. urophylla - P. psidii (Alves et al. 2011) and T. aestivum - P. oryzae [16].

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Decreases in Fv/Fm, Fv'/Fm' , and qP were associated with decreases in ETR (especially in C Leaflets), which is probably related to an excess of reducing power (accumulation of e-) [26,40]. At 12 DAI, the inoculated plants of the susceptible clone responded to excess energy through heat dissipation (C Leaflets), surmised from the fact that there was a significant increase in NPQ at this stage of infection. The results show that, at least at this stage of the infectious process, a low probability that the plant would suffer photoinhibitory damage in the PSII reaction centers.

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A decrease in NPQ was observed during the advanced stages of SALB (> 12 DAI), which would imply the possibility that the plant would suffer photodamage because of an inability to dissipate excess excitation energy [40]. Similar results were described in other species [17]. Alves et al. [15] reported for E. globulus pathosystem - P. psidii that the appearance of chlorotic and necrotic symptoms could occur as a result of oxidative damage, a possibility that might be applicable to this study given the intensification of the severity of infection (> 12 DAI).

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It is likely that photochemical reactions have influenced the CO2 assimilation rate, since aninter-dependent decrease between ETR and A was observed on days when there was greater impact of the disease, especially in the susceptible clone. This behavior contradicted what was reported by Alves et al. [15] and Rios et al. [17] in T. aestivum - P. oryzae and E. globulus - P. psidii in pathosystems, respectively, in which a slight decrease was observed in ETR as compared to the decrease in A. The analysis of physiological parameters (gas exchange and chlorophyll a fluorescence) allow for the following conclusions: (a) photosynthetic rates in clone FX 3864 (susceptible) significantly decreased in response to infection by P. ulei, and decreased by a minor degree in clone FX 4098 (moderately resistant), as a result of decreased stomatal conductance and extrinsic water use in the susceptible clone; (B) the impact on photosynthesis was proportional to the progress and the intensity of the symptoms of the disease, and the phenomenon was intensified in inoculated leaflets during foliar stage 'B'; (c) there is a significant difference in the ability of the photosynthetic apparatus (photosystem II) to capture, use, and dissipate light energy between

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the FX 3864 and FX 4098 clones of H. brasiliensis, evidenced by the photosynthesis of the susceptible clone.

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Acknowledgments

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Financial support for Armando Sterling’s PhD thesis and this research was provided by Colciencias, Instituto Amazónico de Investigaciones Científicas Sinchi, Universidad Nacional de Colombia, Asociación de Reforestadores y Cultivadores de Caucho del Caquetá Asoheca (contract RC No.746 -2011) for the project “Evaluación del asocio agrisilvícola: caucho (Hevea brasiliensis) – nuevos clones de copoazú (Theobroma grandiflorum) mediante el uso de indicadores agronómicos, ecofisiológicos, bioquímicos y epidemiológicos en el departamento del Caquetá-Colombia”.

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Highlights

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SALB severity in clone FX 3864 was significantly higher to that observed in FX 4098. Photosynthesis in clone FX 3864 was affected when infected by P. ulei. A negatives correlation was observed between SALB and all gas exchange parameters. Negatives correlations were observed between SALB and both Fv'/Fm' and Fv/Fm

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