The authors have declared that no competing interests exist.
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
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.
Over the last decade, a plant desiccation epidemic, called
Scortichini et al.
Upon an attack by pathogens or insects, plants can "enlist" the help of protective microorganisms and increase their microbial activity to contrast pathogens
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
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.
This study involved the use of a complex Bio-Fertilizer (BF), which has been defined as “
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.
Seven farms, located near Ugento (LE, Italy), provided access to their olive trees, which age ranged from fifteen to centenary, and were mainly from
# | Farm | Surfaces, ha | |||
C-Control | S-Symbiotic | Treatment date | Monitoring days after inoculation | ||
1 | A | 3.00 | 3.00 | 06-02-18 | 85 |
2 | B | 2.75 | 2.50 | 14-03-18 | 89 |
3 | C | 5.70 | 2.00 | 07-02-18 | 84 |
4 | D | 1.00 | 1.00 | 09-02-18 | 101 |
5 | E | 0.55 | 0.55 | 18-02-18 | 83 |
6 | F | 1.23 | 1.23 | 05-03-18 | 85 |
7 | G | 1.90 | 1.40 | 04-03-18 | 73 |
1 – 7 | A-G | 16.13 | 11.68 | 21-02-18 | 86 |
A commercial symbiotic BF consortium (Micosat F ®, CCS-Aosta,
About 18% of the inoculated trees were evaluated. The morpho-functional features were assessed considering six complex indicators.
A four-point survey card was drawn up (
DSD-Disease SeverityDegree | QBS-Quantity of the Basal Suckers | RMB-Regrowth of the Main Branches | RSB-Regrowth of the Secondary Branches |
0-healty | 0-none | 0-none | 0-none |
1- One dry branch | 1-scarce | 1-short | 1-scarce |
2- two÷five dry branches | 2-low | 2-average | 2-normal |
3 => five dry branches | 3-normal | 3-long | 3-abundant |
4- plant almost dried | 4-abundant | ||
5- plant totally dry |
The codes were processed on the farms using Friedman’s unpaired tests (StatBox V6.5, Grimmersoft, Paris).
According to the indications of previous works
The NIR spectrum of the upper leaf blade was detected using a smart-NIR-SCÏOTM 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 (
S predicted | CS% false positive | SS% |
C predicted | CC% | SC% false negative |
|
C measured | S measured |
A series of leaf composition models were obtained from NIR-SCÏOTM foliar spectra, which had previously been obtained in a sorghum experiment
As described in a recent paper
It was possible to have access to an NIR-SCÏOTM equation, taken from unpublished results of an experimental trial on tomato plants, that provides an indirect estimate of the respiration capacity of the soil (symbol R), measured according to Anderson and Domsch
The results from the bio-fertilizer treated (Symbiotic) and untreated (Control) plots of the farms were elaborated as effective size Ln(S/C) equivalent to (S/C)-1, expressed as a percentage, for the dependent vegetational variables observed in the plants, and for the independent variables measured within the holistic frame. The elaboration gathered the partial results from the main sources of information, namely the differential information from the fingerprints of the leaves and litter-bags, the compositional information from the leaves, and the respiration capacity of the soil. Since there were more variables than the paired groups, the only method available was the Partial Least Squares regression (PLS), provided by StatBox 6.5 (Grimmer Soft, Paris), in which two latent variables were utilized.
The average spectra of the litter-bags from the control plots in the seven groves were calibrated directly with the effective size Ln(S/C) of the plant response to the disease caused by the pathogen from the soil inoculation. For this purpose, the spectra imported into the WinISI II v1.04 chemometric software were math-treated as 2nd derivates (code SNV, 2,8,8,2), and the observed responses were then fitted to spectra using the modified partial least squares (MPLS) method, in which two latent variables were admitted, and the model was cross-validated.
On the basis of the elaboration of the codes carried out on each farm, by means of the unpaired Friedman’s tests reported in
Groves | DSD-Disease Severity Degree | QBS-Quantity of the Basal Suckers | RMBRegrowth of the Main Branches | RSBRegrowth of the Secondary Branches | |||||||||
C,S | d_S/C% |
|
C,S | d_S/C% |
|
C,S | d_S/C% |
|
C,S | d_S/C% |
|
||
A | C | 1.85 |
|
0.95 | 0.73 | 0.95 | |||||||
S | 1.79 | -3% |
|
0.93 | -2% |
|
0.95 | 30% |
|
1.03 | 8% |
|
|
B | C | 1.10 |
|
1.18 |
|
0.79 |
|
0.03 |
|
||||
S | 1.10 | 0.0% |
|
1.00 | -15% |
|
0.64 | -19% |
|
0.10 | 233% |
|
|
C | C | 3.00 |
|
4.42 |
|
3.16 |
|
3.24 |
|
||||
S | 1.79 | -40% |
|
2.50 | - 43% |
|
1.79 | -43% |
|
1.18 | -64% |
|
|
D | C | 3.38 |
|
1.46 |
|
0.62 |
|
0.77 |
|
||||
S | 3.00 | -11% |
|
1.23 | -16% |
|
1.62 | 161% |
|
0.85 | 10% |
|
|
E | C | 1.86 |
|
1.00 |
|
0.62 |
|
1.00 |
|
||||
S | 1.93 | 4% |
|
0.79 | -21% |
|
1.08 | 74% |
|
1.07 | 7% |
|
|
F |
C | 1.30 |
|
3.22 |
|
1.22 |
|
0.91 |
|
||||
S | 3.43 | 164% |
|
1.22 | -62% |
|
0.65 | -47% |
|
0.91 | 0% |
|
|
G | C | 2.78 |
|
0.09 |
|
1.66 |
|
0.81 |
|
||||
S | 3.00 | 8% |
|
0.34 | 278% |
|
1.72 | 4% |
|
0.16 | -80% |
|
|
Means | C | 2.18 |
|
1.76 | 1.26 | 1.10 | |||||||
S | 2.29 | 5.0% |
|
1.14 | -35.0% | 1.21 | -4.0% | 0.76 | -31% |
Farm F was excluded from the final holistic model.
Overall (
S | 46% | 66% |
C | 54% | 34% |
Total | C | S |
Farms | CC | SS |
A | 72% | 79% |
B | 67% | 68% |
C | 67% | 93% |
D | 72% | 72% |
E | 65% | 73% |
F | 65% | 70% |
G | 57% | 61% |
Means | 66% | 74% |
A greater reflectance was observed for the leaves of the Symbiotic olive trees (
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 (
Constituents (X) | Overall Mean | R2 | CV% | Prob. of factors and interaction | Within-farm Contrasts (Y) | ||||||
Farms | BF | Int. | #S+ | #= | #S- | r(X,DSD) |
|||||
Water | % | 70.38 | 0.08 | 3.8 | <.0001 | <.0001 | <.0001 | 4 | 2 | 1 | -0.33 |
ADF | % | 46.48 | 0.07 | 2.2 | 0.0007 | <.0001 | <.0001 | 4 | 3 | 0 | -0.53 |
Non-digestible NDF | % | 28.53 | 0.47 | 2.5 | <.0001 | <.0001 | <.0001 | 3 | 4 | 0 | -0.63 |
ADL | % | 8.96 | 0.19 | 7.0 | <.0001 | 0.0012 | <.0001 | 3 | 2 | 2 | 0.59 |
Ether extract | % | 1.38 | 0.13 | 3.6 | <.0001 | 0.0462 | <.0001 | 3 | 3 | 1 | 0.64 |
Gross Energy | MJ/kg | 17.47 | 0.06 | 0.3 | <.0001 | 0.0788 | <.0001 | 2 | 4 | 1 | 0.70 |
NDF | % | 47.76 | 0.35 | 2.5 | <.0001 | 0.099 | 0.0002 | 2 | 4 | 1 | -0.43 |
Ash | % | 5.76 | 0.18 | 18.1 | <.0001 | 0.9723 | 0.0083 | 2 | 5 | 0 | -0.40 |
NDF digestibility | % | 41.49 | 0.39 | 5.4 | <.0001 | 0.4967 | <.0001 | 2 | 3 | 2 | -0.69 |
Crude fiber | % | 27.17 | 0.05 | 3.0 | <.0001 | 0.5533 | 0.0031 | 1 | 5 | 1 | 0.62 |
Digestible NDF | % | 26.52 | 0.20 | 3.2 | <.0001 | 0.0979 | 0.0767 | 0 | 6 | 1 | -0.41 |
Crop Maturity Index | N | 2.53 | 0.15 | 5.3 | <.0001 | 0.2893 | <.0001 | 0 | 6 | 1 | 0.52 |
Crude Protein | % | 9.32 | 0.20 | 6.2 | <.0001 | 0.0166 | <.0001 | 0 | 6 | 1 | 0.90 |
Total Digestibility | % | 71.43 | 0.37 | 0.7 | <.0001 | <.0001 | 0.0002 | 0 | 5 | 2 | 0.36 |
Hemicellulose | % | 6.41 | 0.22 | 17.0 | <.0001 | <.0001 | 0.0002 | 0 | 4 | 3 | -0.39 |
Free sugars | % | 44.54 | 0.09 | 3.2 | <.0001 | <.0001 | <.0001 | 0 | 4 | 3 | 0.25 |
Cellulose | % | 24.19 | 0.14 | 5.3 | <.0001 | <.0001 | <.0001 | 2 | 1 | 4 | -0.61 |
r(DSD) is the Pearson correlations of the means of six farms (excluding farm F) with the degree of severity disease variation (Y= d_S/C = Ln(S/C) presented in Table 4 for each constituent (X).
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% (
Farms | BF | pH | Prob | H+ | Prob | Water% | Prob | Crude Protein% | Prob | Soil Respiration |
Prob |
LnS/C% | LnS/C% | LnS/C% | LnS/C% | LnS/C% | |||||||
A | C | 5.29 | 0.02 | 5.1 | 0.02 | 70.3 | 0.1553 | 9.53 | 0.5159 | 196 | 0.88 |
S | 5.35 | 1.2% | 4.4 | -14.0% | 70.2 | -0.1% | 9.55 | 0.3% | 194 | -1.0% | |
B | C | 5.17 | 0.44 | 6.8 | 0.44 | 69.8 | <.0001 | 9.42 | 0.1848 | 157 | 0.06 |
S | 5.2 | 0.6% | 6.4 | -7.0% | 70.4 | 0.8% | 9.49 | 0.7% | 139 | -11.3% | |
C | C | 5.56 | 0.47 | 2.8 | 0.47 | 70.0 | <.0001 | 8.98 | <.0001 | 176 | 0.74 |
S | 5.49 | -1.2% | 3.2 | 17.0% | 70.7 | 0.9% | 8.67 | -3.4% | 181 | 3.0% | |
D | C | 5.3 | 0.78 | 5 | 0.78 | 70.8 | 0.9081 | 9.38 | 0.5108 | 184 | 0.48 |
S | 5.28 | -0.4% | 5.2 | 5.0% | 70.8 | 0.0% | 9.32 | -0.7% | 172 | -6.4% | |
E | C | 5.66 | 0.3 | 2.2 | 0.3 | 69.9 | 0.0393 | 9.45 | 0.6595 | 114 | 0.01 |
S | 5.59 | -1.3% | 2.6 | 18.0% | 70.3 | 0.5% | 9.41 | -0.4% | 158 | 37.9% | |
F | C | 5.4 | 0.5 | 4 | 0.5 | 70.8 | 0.0028 | 9.52 | 0.1074 | 183 | 0.66 |
S | 5.43 | 0.7% | 3.7 | -8.0% | 70.4 | -0.6% | 9.40 | -1.2% | 178 | -2.8% | |
G | C | 5.69 | 0.78 | 2.1 | 0.78 | 70.6 | 0.0012 | 9.30 | 0.9169 | 160 | 0.02 |
S | 5.67 | -0.3% | 2.1 | 4.0% | 71.0 | 0.5% | 9.29 | -0.1% | 133 | -17.3% | |
Means | C | 5.44 | 3.7 | 70.3 | 9.37 | 167.2 | |||||
S | 5.43 | -0.1% | 3.7 | 1.0% | 70.5 | 0.3% | 9.31 | -0.7% | 164.9 | -1.3% |
R = Soil respiration (mg Cmic g-1)
The a priori percentage threshold was 50% for CC and for SS. It is shown, in
S | 43% | 70% |
C | 57% | 30% |
Total | C | S |
Farms | CC | SS |
A | 65% | 87% |
B | 63% | 70% |
C | 61% | 85% |
D | 73% | 70% |
E | 100% | 100% |
F | 74% | 74% |
G | 75% | 77% |
Mean | 73% | 80% |
However, higher-levels appeared on individual farms , with an average fingerprint of 80% for SS and of 73% for CC.
The average spectra of the two BF types are displayed in
BF | No. | Reflectance | Standard dev. |
C | 185 | 0.277 | 0.087 |
S | 195 | 0.298 | 0.074 |
S / C % | + 8% | -16% | |
Prob. | 0.0001 |
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%) (
A descending parabolic curve (
Therefore, the symbiotic treatment increased the homogeneity of the leaf.
The litter-bag fingerprint appeared very different from the foliar NIRS fingerprint. In fact, an ascending parabolic trend is shown in
All the variables provided in the study are summarized in
Dependent Variablesobserved in the plants | Independent variables from the analyses X =d_S/C = LN(S/C) | |||||||||||
Foliar NIRS fingerprint | LeafH+ | Soil Respiration (R) | Water | LeafProtein | ||||||||
d_DSD | d_QBS | d_RMB | d_RSB | F_CC | F_SS | d_S/C | L_CC | L_SS | d_S/C | d_S/C | d_S/C | |
A | -3.4 | -2.6 | 31.0 | 7.9 | 72.0 | 79.0 | -14 | 65.0 | 87.0 | -1.0 | -0.1 | 0.3 |
B | 0.0 | -15.2 | -19.4 | 300.0 | 67.0 | 68.0 | -7 | 63.0 | 70.0 | -11.3 | 0.8 | 0.7 |
C | -40.4 | -43.5 | -43.3 | -63.4 | 67.0 | 93.0 | 16 | 61.0 | 85.0 | 3.0 | 0.9 | -3.4 |
D | -11.4 | -15.8 | 162.5 | 10.0 | 72.0 | 72.0 | 5 | 73.0 | 70.0 | -6.4 | 0.0 | -0.7 |
E | 3.8 | -21.4 | 75.0 | 7.1 | 65.0 | 73.0 | 16 | 100.0 | 100.0 | 37.9 | 0.5 | -0.4 |
G | 7.9 | 266.7 | 3.8 | -80.8 | 57.0 | 61.0 | 5 | 75.0 | 77.0 | -17.3 | 0.5 | -0.1 |
Ln(S/C) | DSD-Disease SeverityDegree | QBS-Quantity of theBasalSuckers | RMB-Regrowth of theMainBranches | RSB-Regrowth of theSecondaryBranches |
d_H | -0.155 | 0.131 | 0.215 | -0.378 |
d_R | -0.209 | -0.196 | 0.100 | -0.230 |
F_SS | -0.301 | -0.720 | -0.087 | 0.030 |
L_CC | 0.281 | -0.318 | 0.400 | -0.306 |
Leaf water | -0.133 | 0.130 | -0.699 | 0.400 |
Leaf crude protein | 0.350 | -0.184 | 0.010 | 0.346 |
R2 | 0.96 | 0.48 | 0.72 | 0.59 |
R2 cross-validated | 0.87 | 0 | 0 | 0 |
The mitigation effect on the disease appeared to be highly determined (R2 =0.96), and the model passed the cross-validation process, obtaining an R2 =0.87. There were six characteristic factors:
1) the acidity differential, with standardized factor d_H = -0.155, had a negative sign as the factors were opposite: symbiotic BF lowered the pH, raised the H+ and therefore reduced the disease;
2) the fingerprint of the CC litter-bags (L_CC = +0.281) had a positive sign: when the value was reduced, the pathological degree diminished, a sign that the BF had produced some effects;
3) the fingerprint of the SS leaves (F_SS= -0.301) had a high value and a negative sign: when the value was increased, the disease was reduced;
4) the soil respiration had a favorable negative sign (-0.209): when the respiration increased, the incidence of the disease decreased;
5) the water content of the leaves accounted for -0.133 units, which means that a greater quantity of water flowed and remained in the olive leaves during mitigation and recovery;
6) the crude protein accounted for +0.350 units, the highest contribution to the fitting.
The other dependent variables of the visual appraisal were less predictable. The standardized coefficients in
The attempt to obtain a direct calibration of the average NIR spectra of the Control litter-bags from six farms was successful (
Overall, the soil biota played a key role by provide information and improved insights in this work. There are two environments in nature in which the highest known microbial densities are reached, that is, the human intestine
The response to symbiotic inoculation was first evaluated indirectly, considering the respiratory capacity of the soil in the control and in the inoculated modes; the results of the pairwise comparisons resulted in a negative sign (-0.209), in agreement with a favorable descent of the disease in the holistic model.
Among the agronomic measures of coexistence with
The symbiotic treatment increased the homogeneity of the composition of the S litter-bags, as revealed by the higher fingerprint of the S type which were more prominent than the C types (70%
The close relationships among the average NIR spectra of the litter-bags obtained without BF and the future results expected from a BF inoculation represents a possible extension for this quick method for a rapid soil fertility evaluation: a kind of “
In the holistic model, the fingerprints of the Symbiotic leaves revealed a -0.301 value
Previous research
Accurate disease detection
In the present work, we showed that several spectral plant-trait alterations in the foliar part of the plant (not the canopy) are linked to - or independent of – the mitigation or recruitment of the symptoms. The key mechanisms shown in the present work were reductions in leaf protein, cellulose and hemicellulose, which were balanced by an increase in ADF and lignin, together with an enhanced water content. Although the latter finding may be accounted for by considering a greater fluidity in the xylem vessels and / or increased osmotic pressure in the phloem, which may be attributable to pathogen inactivation, the other variations remain open questions. In the case of grapevines with Pierce’s diseases, symptomatic leaves developed lower water potentials than healthy plants around noon each day, which was related to a reduction in the osmotic potential of the leaf. The discovery that water stress was associated with
In this regard, a recent work
The reinforcement of cell wall parts during the mitigation processes can help protect the leaf from water exchanges. Moreover, along with the progression of disease severity
The protein reduction was in contrast with the expected effects of AM, as observed in a previous study with
According to Lavee and Avidan
An increased concentration of expansins was observed in the vessels of the diseased plants
Contrary to the general expectations about a pH decrease after BF inoculation, and reinforced by our early published result
The rapid methods applied in this experiment do not make it possible to define whether the good results are due to an effective mycorrhization of the roots or to some positive microbial interaction in the rhizosphere with endophytic MOs, whose presence had been verified in the highly complex
Since
Moreover, we have described and tested a set of rapid analyses to monitor the evolution of the disease, not by means of remote sensing, but through friendly contact with the plant and its soil environment. We are conscious that a model with six variables and only six pairwise issued from twelve data-sets cannot be anyway predictive. Thus the original methodological side of this research seems to be that of "learning \ teaching" on simple systems for monitoring the effectiveness of BF: we can anticipate that ongoing researches on various species (maize, tomato, potato) with the same model, are confirming these results in sign and amplitude of the vegetative response, that continues beyond the fruitfulness.
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.