Potency of Commercial Symbiotic Bio-Fertilizer Consortium for Mitigating the Olive Quick Decline Syndrome (OQDS)

The inoculation of soil with a bio-fertilizer (BF), with arbuscular mycorrhiza fungi, characterizes a Symbiotic (S) agriculture mode, aimed at promoting the yield and health of crops through modifications in the rhizosphere as well as in the plant phenotype. The main objective of this study was to reduce the incidence of Olive Quick Decline Syndrome (OQDS, involving Xylella fastidiosa subsp. pauca) that afflicts the olive groves in Apulia (Italy). Non-inoculated control (C) plants were compared with Symbiotic (S) plants inoculated with 20 kg ha of Micosat F ®, through a 15 cm deep scarification, in the groves of seven farms covering an area of 27 ha. In addition to a visual observation of 484 plants, to obtain a gradation of the disease severity, some objective rapid type methods were utilized to survey the plants and soil , namely leaf pH, NIR tomoscopy of the leaves, hay-litter-bag probes coupled with NIR spectroscopy and the prediction of soil induced respiration. The fingerprinting of the S and C types of leaves and litter-bags was ascertained by means of the use of a random forest algorithm in the classification matrices. The results on the symptoms appeared variable: they were significantly mitigated in two groves out of six, but they were aggravated in one. All the rapid measurements became essentials in a “holistic” model which was able to explain over 95% of the average mitigation / null / aggravation response to BF inoculation. The holistic model gathers differential and compositional analyses of the leaf (pH, crude protein, water) and of the soil (respiration), but depends mainly on the fingerprinting of the C and S leaves and litter-bags. Two keys were identified for a successful inoculation: a high degree of variability of the soil conditions permitting hospitality for the BF with enhancement of the microbial activity in the S soil (lowering the fingerprint of the control litter-bags) and homogeneity of the leaves (with increases in the fingerprint of the S leaves treated with BF). In short, the inoculation of diseased plants with one BF consortium is far from being the ultimate remedy to mitigate OQDS in all situations. Further studies are needed, at a field level, to clarify the soil hosting capacity and to define the mycorrhizal and / or endophytic * plant * pathogen interactions, even using rapid methods. DOI: 10.14302/issn.2639-3166.jar-19-2780 Freely Available Online www.openaccesspub.org | JAR CC-license DOI : 10.14302/issn.2639-3166.jar-19-2780 Vol-2 Issue 1 Pg. no. 2 Introduction Over the last decade, a plant desiccation epidemic, called CoDiRO (Olive Quick Decline Syndrome, OQDS), has developed in the olive groves of south Puglia (Italy). One of the main biological factors of this epidemic outbreak has been identified as Xylella fastidiosa subsp. pauca ST53 , a disease that is universally known for having damaged orchards and plants in the Americas . The death of the olive plant follows a process that involves leaf scorching, followed by the death of secondary and primary branches , caused by a prolonged block in the sap flow in the xylem . Scortichini et al. , after a three-year survey, obtained promising results that suggest that an integrated management, which includes regular pruning and soil harrowing, with spring and summer spray treatments with Dentamet® to reduce the severity of X. fastidiosa, may assist in the control of OQDS disease. However, their study scope did not cover the subspecies pauca that is active in Puglia. Upon an attack by pathogens or insects, plants can "enlist" the help of protective microorganisms and increase their microbial activity to contrast pathogens . However, the delivery of a complex Bio-Fertilizer (BF), based on microbial consortia , can act by modifying the plant's physiology and lowering the in-vivo raw leaf pH, which is a concrete and easy endpoint to measure. Apart from accelerating the metabolism, BF acts on the induction of the genes of resistance present in plants, but which are not expressed without prior contact with pathogens. As a result of the inoculum, a consequent activation or suppression of otherwise silent genes is obtained, which recent studies on the genome of plants have identified as being closely related to contrast and alarm activities toward several phyto-pathologies. A demonstration of this was pertaing to the recovery of pears heavily affected by Erwinia amylovora fire blast . On the basis of the above considerations, the objectives of the present work were: i) to revitalize the root microbiome of the infected plants thus reactivating the symbiotic interactions between the root system of the olive tree and the Arbuscular Mycorrhizae Fungi network; ii) to strengthen the defense capabilities of the olive trees by increasing their resilience to the pathogen, through an activation of the latent gene pool; and iii) to evaluate simple and accessible techniques to measure the health status of the olive trees as well as the biological status of the soil. Experimental Procedure This study involved the use of a complex Bio-Fertilizer (BF), which has been defined as “symbiotic” because it contains arbuscular mycorrhizal fungi, spores and propagules, and other microbial species. The use of this BF also falls within the framework of precision agriculture, because the inoculum is distributed precisely in the proximity of the secondary roots of adult olive trees affected by OQDS. After three months, treated Symbiotic (S) and non-inoculated Control (C) plants logged in seven farms were compared to establish their disease severity, by means of a visual appraisal coupled with rapid new tests, namely: litter-bags (differential and respiratory), foliar NIR scanning (differential and compositional) and foliar pH. A final holistic elaboration gathered all the available results from the three main information tools, concerning the plant-soil-BF interactions, in a model that Corresponding author: Giorgio Masoero, Accademia di Agricoltura di Torino, Via A. Doria 10, 10123, Torino, Italy. E-mail: giorgioxmasoero@gmail.com Running title: Mitigation of OQDS (CoDiRO) by means of a bio-fertilizer


Introduction
Over the last decade, a plant desiccation epidemic, called CoDiRO (Olive Quick Decline Syndrome, OQDS), has developed in the olive groves of south Puglia (Italy) 1,2 . One of the main biological factors of this epidemic outbreak has been identified as Xylella fastidiosa subsp. pauca ST53 3,4,5 , a disease that is universally known for having damaged orchards and plants in the Americas 6,7 . The death of the olive plant follows a process that involves leaf scorching, followed by the death of secondary and primary branches 8 , caused by a prolonged block in the sap flow in the xylem 9 .
Scortichini et al. 10 , after a three-year survey, obtained promising results that suggest that an integrated management, which includes regular pruning and soil harrowing, with spring and summer spray treatments with Dentamet® to reduce the severity of X. fastidiosa, may assist in the control of OQDS disease. However, their study scope did not cover the subspecies pauca that is active in Puglia.
Upon an attack by pathogens or insects, plants can "enlist" the help of protective microorganisms and increase their microbial activity to contrast pathogens 11 . However, the delivery of a complex Bio-Fertilizer (BF), based on microbial consortia 12

Experimental Procedure
This study involved the use of a complex Bio-Fertilizer (BF), which has been defined as "symbiotic" because it contains arbuscular mycorrhizal fungi, spores and propagules, and other microbial species. The use of this BF also falls within the framework of precision agriculture, because the inoculum is distributed precisely in the proximity of the secondary roots of adult olive trees affected by OQDS.
After three months, treated Symbiotic (S) and non-inoculated Control (C) plants logged in seven farms were compared to establish their disease severity, by means of a visual appraisal coupled with rapid new tests, namely: litter-bags (differential and respiratory), foliar NIR scanning (differential and compositional) and foliar pH.
A final holistic elaboration gathered all the available results from the three main information tools, concerning the plant-soil-BF interactions, in a model that was there after used to explain a possible symbiotic mitigation process of the disease.

Material and Methods
Farms and Groves (Table 1) Seven farms, located near Ugento (LE, Italy), provided access to their olive trees, which age ranged from fifteen to centenary, and were mainly from cv. Ogliarola di Lecce, although a few were also from cv. Leccino (Table. 1).

Bio-Fertilizer
A commercial symbiotic BF consortium (Micosat F ®, CCS-Aosta, Figure 1) was used at a dose of 20 kg ha -1 . The root inoculation was a 15 cm deep scarification in which the microbial fertilizer was distributed. A furrow ( Figure 1), which allows the surface roots of the plants to be inoculated, was made close to the trees. 1

1-Visual Evaluation
A four-point survey card was drawn up (Table. 2) The codes were processed on the farms using Friedman's unpaired tests (StatBox V6.5, Grimmersoft, Paris).

2-Foliar pH
According to the indications of previous works 12,14,15 , the in vivo raw leaf pH measurements were carried out using a BORMAC pH-meter "XS pH and not to a decrease in pH.

3-Foliar NIRS and Discrimination
The NIR spectrum of the upper leaf blade was detected using a smart-NIR-SCÏO TM spectrometer (Consumer-Physics, Tel Aviv), which operates in the NIR 740-1070 nm band. The chemometric elaborations were carried out by means of the SCÏO-Lab software, which operates using AKA (As Known As) recognition matrices, built by means of a Random-Forest algorithm, and provides a percentage of recognition of the cells of the matrix (Table 3).

4-Foliar NIRS and Composition
A series of leaf composition models were obtained from NIR-SCÏO TM foliar spectra, which had previously been obtained in a sorghum experiment 15 .
The equations were applied to the olive leaf spectra to obtain an estimate of several constituents. The predicted values were analysed by means of a bi-factorial GLM linear model (SAS 9.1).

5-Litter-Bags and NIRS for Discrimination.
As described in a recent paper 16    it was possible to measure the aerobic activity, induced by the microbial consortium, using the infrared meter.
The correlation between the estimated and measured data resulted to be sufficiently high (R 2 cross-validated 0.86) to be considered reliable under comparable conditions.

A Holistic Model
The results from the bio-fertilizer treated  Table 4 Table 4, the degree of disease severity was significantly reduced in two cases:  Foliar NIRS-Fingerprint of the Symbiotic Treatment (S) and of the Control (C)

Plant Disease and Vegetation
Overall (Table 5), the S leaves were recognized as S at 66% (P <0.0001), a significantly greater value than the 54% found in the controls, which, however, was still statistically significant as there were numerous samples. However, higher-level fingerprints appeared on individual farms, with an average 74% for SS and 66% for CC.

Foliar NIRS-Changes in Spectra
A greater reflectance was observed for the leaves of the Symbiotic olive trees ( Figure 3).

Foliar NIRS-Changes in Leaf Composition and Correlation with the Disease Severity Degree
In addition to a physical difference, a chemical variation also appeared in the leaves three months after the treatment. Their chemical composition resulted differentiated for all of the seventeen considered traits ( with the variation in the disease severity degree (last column in Table 6), as clearly shown in Figure 4, where a positive relationships linked the two traits, which means that a decrease in the Ln(S/C) of protein favored a reduction in the disease.

Foliar pH
The pH was only significantly modified by the BF treatment on farm A, where it increased by 1.2%. In two other cases, it increased by 0.6 -0.7%, while it was on average acidified by 0.8% (Table 7) in four cases.
Litter-Bag-NIRS Fingerprinting of the Symbiotic Treatment (S) and of the Control (C) The a priori percentage threshold was 50% for CC and for SS. It is shown, in

NIR Spectra of the Litter-Bags
The average spectra of the two BF types are displayed in Figure 5. It appears that a greater reflectance of radiation distinguished the S litter-bags.
The average S/C ratio increase is constant at around +   The a priori percentage was 50% for CC and for SS.    Table 4 for each constituent (X).  (Table 9). On the other hand, the standard deviation of the S spectra is reduced by 16%, compared with the corresponding C, and this is a sign of greater homogeneity. Figure 6 shows the two trends of the upper level and a minor dispersion pertaining the S spectra.

Respiration Capacity of the Soil (R) Estimated by Means of the NIRS of the Litter-Bags
The respiration capacity of the soil, as estimated by means of the NIRS of the litter-bags, increased significantly after the symbiotic treatment at the roots and in the soil on farm E (+ 38%), while it appeared somewhat decreased on G (-17%) and B (-11%) ( Table   7).

Discussion
The Soil Overall, the soil biota played a key role by provide information and improved insights in this work.        Prob. 0.0001 Table 9. Average reflectance of the litter-bags.   There are two environments in nature in which the highest known microbial densities are reached, that is, the human intestine 18 and plant roots 11 (1 x 10 13 and 10 11 per ml/g, respectively) 19

The Leaves
In the holistic model, the fingerprints of the According to Lavee and Avidan 30   In substance, the inoculation of BF consortia is far from being a definitive remedy for OQDS, and it needs further investigations about the hosting capacity of the soil and concerning mycorrhizal and / or endophytic * plant * pathogen interactions, even using rapid methods (litter-bags, foliar pH, NIR tomoscopy) in field studies. However, some indications now exist on how to fight the disease with weapons that are to some extent more environment friendly and sustainable.