The Raw pH in Plants: A Multifaceted Parameter

The measurement of the in vivo raw pH of vegetative organs is a unusual way obtaining plant knowledge. The authenticity of the pH parameter of the leaf and its independence from soil pH has already been highlighted. In the present work we observe how and to what extent water-temperature mechanisms as well as bio-fertilizers inocula can affect the raw pH and how great the biodiversity is in plants. A trial with Arabidopsis thaliana in a phytotrone has shown that, in the dark, the raw pH did not change from +18 to +35 °C (b = -0.0027 N.S.), while in the light, the regression coefficients were significant and negative, and the acidification in the leaves progressed from high (-0.0097) to normal (-0.0127) and then to low (-0.0370) water level. We have confirmed that warming induces a decrease of raw petiole pH of -0.070 pH C in grapevine leaves. In accordance with water-temperature mechanisms, the raw pH in grapevines has been found to be significantly higher in well-watered plants (pH = 4.29) than in stressed ones (4.12), with a pH decay of -3.9%. On the other hand, an average reduction of 0.10 units of raw pH would signal an increase in water stress of about -0.59 Mpa. Among the phenomena that can influence the raw pH, we have outlined three biotic factors: i) acidification as a result of a symbiotic farming fertilization i.e through the use of mycorrhizal and microbial fertilizers, with an average decay of around -3%, as a probable signature of symbiosis; ii) an “acida plantarum natura” scenario over 49 species, ranging from pH 3.06 to 6.38 ; iii) a strong (R = 0.9) inverse polynomial pseudo-relationship of the number of fungicide sprays on the raw pH in a set of 15 species. It is suggested that this simple new multifaceted parameter can deserve interest. DOI :10.14302/issn.2639-3166.jar-18-2397 Correspondinguthor: Giorgio Masoero, Accademia di Agricoltura di Torino; Torino, Italy, Email: Giorgioxmasoero@gmail.com Running Title: Raw pH, a multifaceted parameter


Introduction
The in vivo raw pH of vegetative organs is a unusual measurement in plant knowledge, that almost sounds like a paradox when compared to the resonant importance of soil pH 1 or pH and Eh 2 and moreover of pH and Eh in hydroponics systems 3,4 . In fact the raw mass pH was an historic pivotal keystone, since AI Virtanen, NP in 1945, and still it is worldwide, everywhere silage fodder is adopted: in truth the present study is intended to deep inside the in vivo raw pH in crops and in orchards, that is preand not post-mortem phase. It must be pointed out that in the most studied vegetal species, Arabidopsis thaliana, the pH has been deeply discerned among intracellular components 5 but the raw foliar pH, intended as the raw matrix of leaf tissues, remains neglected. In the framework of a study on mycorrhizal and microbial effects on maize 6 a systematic significant response emerged in the in vivo raw pH of maize stems, showing an acidification de-gradient from roots (pH -7% in mychorrized corn) to stem pH at ears height (-4%).
After this first results, preliminary surveys in grapevine ascertained that the Flavescence dorée, a phytoplasma diffused in Latin EU-area 7 attack determined an elevation of the pH in unhealthy sub-branch and petioles 8 . Raw pH fall-out in Sorghum sudanensis leaves has been recently recognised as a sign of mycorrhizal inoculation 11 . So far, a relevant result was the appearance of acidic nature in the raw tissues of corn and grapevine that were lower than the previous leaf pH values determinated in 92 species from the Cornelissen team 9,10 .

Experimental Procedure
The aims of the present study were to pursuit the investigation of this paradox multifaceted parameter with address for either: i) a multi-species variation, and ii) a dependency on the aerial temperature and on the plant water status that, iii) a different susceptibility to fungal and or bacterial diseases. Four bodies of data were considered for the setup of the present work.
Further comparative elaborations and suggested hypothesis are reported in the discussion.

Measuring Raw pH
The in-vivo raw pH measurements were conducted using a BORMAC "XS pH 70" pH meter (www.giorgiobormac.com), range pH 0÷14, two decimals, provided with a combined plastic-glass electrode Hamilton Peek Double-PoreF, / Knick, dimensions (LxØ) mm 35×6, terminating with a very small and sensible tip sensor; other types were unstable and unreliable. The insertion of the tip in the petiole was facilitated by a small drill fitted with a 2 mm bit. In total 4181 leaves were examined for raw pH in the petiole axis, basal side.
When the petiole was too thin for a tip insertion (olive, grape) then an axial cut by a lancet was executed, and the sensitive tip was wrapped by the two wings of petiole fork. For the Arabidopsis thaliana examination, several small packages of leaf blades were pierced with the tip. The maize pH was measured in the stem at the middle of 2 nd internode 6 and not in a part of the leaf.

Multispecies Variability
As a total forty-nine species (Table 1,Table 2) were sampled from twenty-nine botanic families, relevant to several orchards, trees, vegetables and ornamental species. The grapevine was the most represented species (No. 2190) suited by the maize (792). Geographic origins were Piedmont (vine, vorn and several other), Emilia (hazelnut, blueberry, cherry and kiwi) and Puglia (olive). The pH values were analysed according the species, by a one-way ANOVA, using PROC GLM, Tukey's HSD, by SAS V. 9 software (SAS Institute, Cary, NC, USA).

Temperature Timeline Effects
Four Barbera grapevines were grown in-pot outdoor at the DISAFA experimental implant (45°03'58.6"N 7°35'23.8"E). The pH was measured on fifty-six leaves, without automatic correction for temperature, and sampled at three different timepoints (8-9; 12-13 and 19-20) during October 2016. In this part of the study were comprised other raw pH measurements recorded on Barbera grapevines from the same implant and sampled in July on the late morning.
The external temperature was considered as the independent variable in a regression study of pH. The hour of the day was also plotted to consider the daily In the report published from the EFSA EU-ERA network 12 , in the original Table 17 12 were exposed the frequencies of the fungicide and insecticide sprays for 13 orchards and crops. Because no data were available for pear, a key species for pH involvement, and coffee, some specialist colleagues were asked about. These mean values were colligated with the mean pH of the species by using polynomial models. A special study concerned the differential outbreaks of the fire blight (Erwinia amylovora) in apple and pear, as meticulously monitored in the Bolzano Province 13 from 1999 to 2016 in some 8.185 farms; the relative incidence and odds ratio referred to farms and hectares.

Multispecies Variability
The raw pH appeared as a very wide-distributed parameter across the species (Figure 1) An apparent scenario of acidity, ( Table 1,

Temperature and Water-Stress Effects
In the experiment with grapevine aimed to an assessment of the pH to the aerial temperature, it was observed a saddle trend from morning to evening ( Figure 2). The value was stable overnight and it depended on the combination between sunlight and aerial temperature. Otherwise during the dark period the pH remained stable. Such rise of acidity in the middle day can be ascribed both to a thermal factor or to a sun irradiance available abundance.
Where regressing the pH and the aerial temperature ( Figure 3) the two variables appeared as     Table 3).
The raw petiole pH was also sensible to divergent conditions in the soil moisture. In the pot experiment, with grapevines from Grenache vineyard (Table 4 and Figure 4) the pH was significantly different in the well watered (pH = 4.29) and in the stressed conditions (4.12). The pH decay was 3.9%, but in terms of [H + ] the increase was 47%.
Where looking at the individual values (Table 5) the relationships direct and inverse appeared significant Mpa.
The experiment with Arabidopsis thaliana in a phytotrone raised highly significant level (Table 6) showing that in the dark the raw pH did not change in the interval from +18 to +35 °C (b = -0.0027 N.S.; Figure 5; Table 7) while under the light, the regression coefficients were significant and negative, progressing in acidification from a water levels high (-0.0097), to a normal (-0.0127), and to a low (-0.0370) .

Symbiotic (Mycorrhizal and Microbial) Effects
The main factors were significant, with the two factor interaction near significant (Table 8). With reference to Table 9 (Table 10).
When looking at the fungal pest occurrences a strong inverse polynomial relationship (R2 = 0.9) was interpolated among the mean pH of 15 species and the number of fungicide sprays (Table 11 and Figure 6).
According the Authors, alkalization process seems to have the general goal to minimize and obstruct fungal growth biochemically.
In a broad scenario of plants the pH of phloem exudate is characteristically alkaline (pH 8.0 to 8.5) and belong from non-reducing sugars (sucrose), amides (glutamine and asparagine), amino acids (glutamate and aspartate) and organic acids 19 . Acidity of the whole green tissues has longely been utilized in silage operations; methods adopted to improve silage feeding value include rapid wilting and acidification, either by acids products or use of anaerobic inoculants 20 ; however the pH of the raw material is not considered, at most the buffering power is taken into account.
The team of Cornelissen 9,10 elicited the leaf pH as a new plant trait and explored it as featuring in the carbon or nutrient cycling context in the framework of 92 species (Table 11) which quintile distribution appears quite similar to that of the present 49 species but discarded towards less acidic values (Figure 7).
The authenticity of the pH parameter has been highlighted 10 , upsetting the myth of the soil pH: as shown in Figure 8 in fresh leaves the leaf pH will rise only of 0.036 pH soil -1 (about 0.7%) but in pre-dried herbages the inverse occurred (-0.4%). Important to note the differences in pHs: after pre-drying at 60°C for 48 h and reidratatying, the increase of pH was about 4.3%. This confirm the sense of the relationships pH-temperature-humidity (Table 13).
As to the positive relationships between the pH and the water availability a primary apparent sign of stress is the rise in leaf temperature, an infrared sensed measurement basic for plant-water relations, and specifically for stomatal conductance monitoring 21 . In the present work we have commensurate the drop in petiole raw pH to a water stress condition, but chiefly the linear drop was accounted for by the increase of aerial temperature (-0.07 pH °C -1 ); in fact in the equations C (Table 3) when the quadratic term was considered, the fit did not improve. It is well known that when the temperature roses over 25°C, the pH declines in force of the normal autoprotolysis -autoionization of the water.
But it should be pointed out that the acidic solutions are affected less by this phenomenon than the alkaline ones, and especially when natural buffers are present.
In     Faced with a multifaceted nature of the raw pH acidic wide range, it is possible to ask which lowest common denominator (a large entity) or which greatest common divisor (a small entity) should be considered?
In the authors' opinion, assuming the pH as a common divisor, in the pseudo-relationships NFS / pH ( Figure 6) a common denominator is the propensity towards fungal attack when the pH is more acidic, with vinegrape and apple as driving species, but also the propensity towards bacterial attack when the pH is less acidic, where a clear demonstration outcomes from a balance among the apple vs. pear diseases (Table 12). Maize is a very acidic species; in Table 1 it is positioned as 8th /49, but it is excluded from the Table 10  In perspective, the warming from climate change scenarios, could affect a rise in raw acidity of plants, fearing that fungicide sprays could be increased by one per C° degree rise. In the Figure 9 we have hypothesized a very high increase of +1.8°C in the average temperature; the major incidences as percentage of NFS increase, reside in the median 5.5 ÷ 5.9 pH range.
The rise in aerial temperature will grew fungal as well as bacterial disease pressure. In a grapevine district Salinari et al. 29 have calculated that two more fungicide sprays were necessary under the most negative climate scenario, compared with present management regimes. The increase of presumed 1.8 C° will correspond in the present paper (Table 3,

Conclusion
At precision farming operational plan, the raw pH could be considered as metabolic signal of water stress; moreover, vibrational spectroscopy in NIR range could be correlated with it beyond the thermal infra-red (IR) signature.
Symbiotic farming is a sustainable and resilient ausilium to preserve and improve soil fertility, where the raw pH could be a response sign of efficiency for microbial biota evolution in the rhizosphere.
In perspective, the warming of plants from climate change scenarios, could push a rise in plant acidity, fearing that fungicide sprays could be increased in future, moreover when considering the consequences from a strong negative impact of the water stress on the raw pH.
A suggestive paradoxal challenge is given by the acidifying reaction of arbuscular mycorrhizae which instead of favouring the conditions for development of