Control of Mal secco disease in lemon by drip irrigation of fungicide

doi:10.21203/rs.3.rs-3787339/v1

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Control of Mal secco disease in lemon by drip irrigation of fungicide

https://doi.org/10.21203/rs.3.rs-3787339/v1

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published 02 Aug, 2024

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The ‘mal secco’ disease of citrus is caused by the mitosporic ascomycete fungus Plenodomus tracheiphilus (formerly Phoma tracheiphilus (Petri)). Mal secco of citrus is a highly destructive vascular disease of lemon and other citrus, presently confined to the Mediterranean basin, which has a relevant economic impact on the citrus industry. The fungal pathogen infects the host tree by penetrating through wounds in the roots or the canopy. Canopy infection spreads quickly into the main branches and trunk and tree mortality usually occurs. The most typical symptoms are venial chlorosis, leaf wilt, red coloration of the xylem and die back of twigs and branches. Current acceptable control of the disease is mainly by sanitation of infected wood, and cooper application during the winter, to prevent germinating spores from infecting the plant. No effective chemical control was reported for the control of the disease. Here we report the development of a drip irrigation protocol for the protection of trees and the control of the disease by applying Futriafol 125 gr/liter, five times during the year. The progress of the disease symptoms in the treated trees is inhibited dramatically allowing the sanitation process to become easier, significantly shorter to perform, and cheaper than in untreated trees. The longer the orchard is treated, the less symptoms of the disease are diagnosed in the trees. We have treated orchards for more than six years and established a commercial protocol for the growers, helping them to control the disease.

Lemon

fungal disease

control

irrigation

Mal secco disease caused by Plenodomus tracheiphilus (formaly Phoma tracheiphila) (Petri) Kanc. & Gik. is a devastating disease of citrus in the Mediterranean basin, around the Black Sea and in Asia Minor (EPPO and CABI 1990; Palm 1987; Solel 1976; Timmer et al. 1988). Almost all citrus species are susceptible to P. tracheiphilus if inoculated artificially. In the field, the disease is highly destructive mainly to lemons (Citrus lemon), citron (C. medica), and lime (C. auratifolia) (Graniti and Perrota. 1988). The fungus and the disease have been studied and documented extensively in studies that have focused on epidemiology, the toxins involved in the pathogenicity of the fungus and possible methods of disease management (Chorin and Chorin 1956; Fogliano et al. 1998; Graniti and Perrota 1988; Migheli et al. 2009; Nicasio et al. 2000; Palm 1987; Rotem et al. 1998; Solel 1976; Solel et al. 1972; Timmer et al. 1988).

In Israel, the pathogen usually infects the host tree by penetrating through wounds in leaves and branches (Horin et al. 1966). Typical symptoms include chlorosis of leaf veins, leaf wilt, red coloration of the xylem and die-back of twigs and branches. The pathogen proceeds slowly downward from the infected young shoots to the branches and main limbs; when the trunk and roots are infected, the tree dies. This movement is aided by phialoconidia, which are translocated in the xylem via the plant’s transpiration flow (Palm 1987; Timmer et al. 1988). When infection occurs through the roots, the sudden death of the tree, caused by the rapid infection of the trunk, is typically observed (Graniti and Perrotta 1988).

Control of the disease is mainly based on sanitation of diseased plant tissue accomplished by cutting and burning of dry and symptomatic branches and trees. This method is time consuming and very expensive in most countries were the disease is established. Copper based chemicals are applied during the winter in an effort to control the germinating spores of the fungus. Spores are spread by rain and wind in the winter. Yet, due to the very low survival of these compounds on the canopy during rain, this method of control is considered ineffective (personal knowledge). Application of other chemicals was never proved effective, probably due to the location of the pathogen in the xylem, protecting it from the chemicals when in the plant. In the case of woody plants, systemic chemical products are not effective in the woody tissue and may give some effect when applied on leaves and wonds (Wu et al. 2020). The idea of drenching chemicals to trees is not new (Mondello et al 2018) but was never proved effective in the case of mal secco or as a control method for fungal pathogens of citrus.

In this study we report the development of a drip irrigation protocol of chemical control product, for the protection of trees and the control of the mal secco disease in lemons.

Lemon groves, type and location.

During the years 2016-2017 an experiment for the evaluation of chemicals for their possible influence on disease development by drip irrigation was set in a one hectare, tree year's old lemon grove located in the village of Oza at the north east of the Negev desert in Israel. The lemon grove was designed with its eastern half part (0.5 hectare) of Vilafranka veriety grafted on volka mariana rootstock and the western half part with Vilafranka on Macrophylla rootstock. The western part was chosen for the experiment as a large number of the trees in this part expressed mal secco symptoms while the trees on the eastern part grafted on volka mariana rootstock did not express symptoms.

The experiments plot had 419 trees when planted (2014). By the second year (2016) about 30% of the trees were uprooted due to mal secco. During 2017 we planted 128 new trees Vilafranca grafted on volka mariana, filling for dead uprooted trees. The grove had 15 rows with 26 to 29 trees in each row. Each row served as a repeat for a treatment. There were tree repeats (tree rows) for each treatment. During 2016 we tested the influence of 4 different chemical products, by applying them 6 times, one month apart, from September to October (two times) and from March to May (tree times).

Concentration and timing of applications

During the year of 2017 a concentration experiment was designed using the most effective product of the 4 tested. Five treatments were applied to the Oza orchard:

Water control – untreated, "Hosen" (Futriafol 125 gr/liter, Adama, Israel) 2 liter/ hectare (2^nd year of application), Hosen 2 liter/ hectare, Hosen 1 liter/ hectare, Hosen 4 liter/ hectare

The treatments were applied five times at October and November (one month apart) and March (right after the first spring irrigation of the orchard), April and May (one month apart). The product was applied by injection into the dripping system using a pump. The product was added only after a short irrigation of the trees (about 30% of the amount given to the orchard at this time of the year). At the end of Hosen application, the trees were further irrigated with 50 cubic meters of water per hectare.

Each treatment was applied to three rows of trees with 26 to 29 trees in each. Each row was considered as a repeats of the treatment. Rows used as untreated control and rows treated with Hosen 2 liter/ hectare on year 2016 were used on year 2017 for the same treatment for the examination of accumulated effect of the treatment. Rows used during 2016, for other un effecting products, were used for the other concentration treatments. The same design and treatments were continued during the seasons of 2017-2018 and 2018-2019.

Mal secco symptoms evaluation

Mal secco symptoms were evaluated by counting the number of new dry twigs and branches displaying at least two additional symptoms as leaf fall, petioles left on the branch or xylem orange-pink coloration. Evaluation was done every two weeks from the beginning of April to the end of Jun.

All Trees in the experiment were subjected to sanitation by pruning and removal of infected branches and were "cleaned" from symptoms prior to treatment. No further sanitation was applied to trees in the experiment during the years of the experiments in order to enable the evaluation of new disease symptoms (new dry branches).

The most effective product of the four tested during 2016-2017 was "Hosen" (Futriafol 125 gr/liter, Adama, Israel), applied at 2 liter/ hectare (results not presented). During 2017-2018 we treated the trees with Hosen at different concentrations to find the lowest and most effective concentration that could decrease mal secco symptoms in the trees.

Mal secco symptoms appears in Israel during the beginning of the spring when the trees start blooming around the beginning of April to mid Jun when the temperatures rise and the development of the disease is reduces. We evaluated mal secco new symptoms appetence seven times, every approximately two weeks from mid April to the end of June (table 1).

Table 1. Evaluation of new mal secco symptoms in the Oza orchard in the year 2018 (2017-2018)

Treatment liter/ hectare	Percentage of trees with new symptom- Cumulative result
Treatment liter/ hectare	17.4.18	3.5.18	15.5.18	28.5.18	8.6.18	20.6.18	1.7.18
Control	1.65a	8.35 a	10.00 a	11.65 a	15.00 a	16.65 a	20.00 a
Hosen 2 l/h (year II)	0.00 a	0.00 a	0.00 a	0.00 b	0.00 b	0.00 b	0.00 b
Hosen 1 l/h (year I)	0.00 a	3.41 a	3.41 a	3.41 ab	3.41 ab	5.11 ab	6.76 ab
Hosen 2 l/h (year I)	0.00 a	0.00 a	0.00 a	1.77 ab	3.54 ab	5.31 ab	5.36 ab
Hosen 4 l/h (year I)	0.00 a	0.00 a	0.00 a	0.00 b	0.00 b	0.00 b	0.00 b

Results presented as percentage of the infected trees out of all living trees in the treatment. Same letters represent statistical similarity according to SNK, P<0.05

At the end of the evaluation, we noticed that at the beginning of the spring the symptoms on the trees are fewer. Closer to the end of the spring (end of June) we could see more trees expressing symptoms. The trees treated with Hosen 2 l/h (year II) for two successive years did not display symptoms while on the Hosen 2 l/h (year I) trees we could see symptoms. Hosen 1 l/h (year I) treatment was the less effective and while Hosen 4 l/h (year I) was effective as Hosen 2 l/h (year II).

Table 2. Evaluation of new mal secco symptoms in the Oza orchard in the year 2019 (2018-2019)

Treatment liter/ hectare	Percentage of symptomatic trees - Cumulative result
Treatment liter/ hectare	9.4.19	10.5.19	24.5.19	6.6.19	30.6.19
Control	20.00 a	46.67 a	68.33 a	68.33 a	71.67 a
Hosen 2 l/h (year III)	0.00 b	0.00 c	4.92 bc	6.51 bc	6.51 bc
Hosen 1 l/h (year II)	1.52 b	16.96 b	21.93 b	27.36 b	30.84 b
Hosen 2 l/h (year II)	6.95 ab	8.89 bc	12.22 bc	15.56 bc	19.18 bc
Hosen 4 l/h (year II)	2.56 b	2.56 bc	2.56 bc	2.56 bc	2.56 bc

Results presented as percentage of the infected trees out of all living trees in the treatment. Same letters represent statistical similarity according to SNK, P<0.05

We applied the same treatments on the same trees during the second year of the experiment (2018-2019). The same results were found for the different treatments at the end of the evaluation of the second year (table 2). At the end of the second year we found that 71.67 percent of the untreated control trees were displaying mal secco disease symptoms. The Hosen 2 l/h (year III) treatment had only 6.51 percent of the trees with symptoms. Hosen 2 l/h (year II) had higher percentage of trees with symptoms (19.18) yet ,still much lower than the control. Hosen 1 l/h (year II) as in the first year reduced the percentage of symptomatic trees but not as well as Hosen 2 l/h (year II or III) (30.84%). The most effective treatment was again Hosen 4 l/h (year II) with only 2.56% of the trees with disease symptoms.

Following these results, three more infected orchards, two lemon orchards in the Negev desert (Patish and Paame Tashaz, south of Israel) and one lime orchard in the Golan Heights (Gonen, north of Israel) were treated with "Hosen" with the different concentrations. All experiments were conducted for at least two years. Application of Hosen at the spring and autumn (five applications – March, April and May and October, November). These orchards differ from the Oza orchard by their age, the Oza orchard was a young orchard (3 years from planting at the beginning of the experiment) with relatively small trees in size, while the other three orchards were older orchards (6 to 9 years old at the beginning of the experiments) with bigger trees in size. Summary of the results are presented in table 3.

Table 3. Evaluation of new mal secco symptoms in the Patish, Paame Tashaz and Gonen orchards in the year 2019 (2018-2019).

Treatment liter/ hectare	Percentage of symptomatic trees compered to untreated control at the end of the experiment
	Patish	Paame Tashaz	Gonen	Oza
Control	100	100	100	100
Hosen 1 l/h (year II)	64.5	80.7	77.5	44.3
Hosen 2 l/h (year II)	53	39.7	41.3	18.8
Hosen 4 l/h (year II)	26.8	18.1	36.7	0.0

Results are given as percentage of trees displaying symptoms compared to the number of symptomatic trees in the relevant control

In the past, attempts to control mal secco disease of citrus by injecting chemical control products directly into the xylem (Solel et al. 1972) or applying the compounds directly to the roots of the tree by pouring it onto the soil were done (personal knowledge, unreported results). As part of a large project we had in Israel (2014–2019), trying to find new approaches for the control of mal secco in citrus and mainly in lemon and lime, we tried application of chemical control by the irrigation system. The logic was that the slow application by the water would influence the ability of the active ingredients in penetrating and migrating in the trees tissue. Prior to the first year of the orchard experiments, we tested different fungicides commercial products in the laboratory in in vitro petri plate experiments and found the most active products against the pathogen. During the first year, we tested these products activity in the orchards and chose to continue in the second and third years experiment, with only one product, the one we found to be the most effective in reducing the disease symptoms in the orchard. At the second year, we expended the research by adding three more orchards to the experiment in three different locations in order to test the treatments under different conditions including location of orchards, type of citrus and age and size of the trees. Analyzing the results from all four orchards in the experiment, we concluded that the size of the trees and the severity of infection were critical for the success of the treatment. As lower the infection severity and smaller in size the trees were at the beginning of the application, the better the control of the disease symptoms are. We noticed that the treatment was much more efficient when applied to trees infected by up to 30% of their volume. We recommend trimming the trees to up to 2.5 miters in height; this will allow better and easier sanitation of dry and infected branches. Trees with more than 30% infection should be uprooted and remove from the orchard as it is very difficult to cure the trees from the disease and they serve as inoculum of the pathogen in infecting other trees in the orchard. It is recommended to start applying Hosen 2 l/h as soon as the first symptoms of the disease are diagnosed in the young orchard. Our experience is that in most cases when the pathogen is already established in the orchard it may take months to years for the symptoms to appear on other trees. In many cases, this event occurs simultaneously in many trees in the orchard. We found that there is an accumulated effect of the treatment. As longer as the treatment is applied to the orchards (more years of treatment), the less sanitation of dry symptomatic branches is needed and we see less trees dying from the disease. Sanitation of dry symptomatic branches should be performed by inspection of the orchard once a month and preferably every 2 weeks during the spring (March to end of June) and autumn (October to December) when conditions for symptoms development are favorable. Pruning of symptomatic branches should be to at least one hundred centimeters (one meter) below the symptom of the disease (dead wood, pink-orange coloration of the xylem). We found that continuance application of Hosen 2 l/h five times during the year reduces symptoms significantly more than Hosen 1 l/h (Tables 1–3). Although Hosen 4 l/h treatment substantially reduced disease symptoms compered even to Hosen 2 l/h treatment, we experienced at the first year of application in Oza orchard some phytotoxicity, expressed as leaves edge dry out. This phytotoxicity disappeared as new leaves grow. We assume this phytotoxicity was due to application of the higher concentration at the first irrigation of the trees after the winter, when the upper layer of the soil is dry. We did not see the phytotoxicity if the orchard was first irrigated followed by Hosen 4 l/h application. We did notice in the latter case a minor delay in blooming that disappeared completely at our second evaluation of disease symptoms in June.

The Oza orchard served us as the main experimental orchard as it was the first to be treated and the one in which the treatments were given for the longest period. The other three orchards were older and less homogeneous due to disease severity, size of trees and the ability to perform sanitation of the trees as we recommend. Even though, we can see in Table 3 that the treatment of Hosen 2 l/h influenced symptoms to 40–53% of the number of trees displaying symptoms in the same orchard in the un treated rows. This means that although the condition in the orchards were not optimal for the chemical control we still see positive influence of the Hosen 2 l/h on disease symptoms.

Following the results from the orchards in the experiments, we continued for two more years in a "model orchards" observations system. "Model orchards" are commercial lemon orchards treated by the owners under the acceptable methods with the addition of our mal secco disease treatment. The model orchards were treated by Hosen 2 l/h five times a year as described. At the end of each season, evaluation of disease symptoms including number of trimmed branches along the year, number of dead trees and crop assessment (weight of fruit picked from 10 trees, from 5 different treated rows altogether 50 trees, compared to same number of trees from un treated rows). We found, as we expected, reduction in the number of new symptomatic branches and dead trees in the treated trees compared to former years and untreated trees. We found no significant differences in crop weight evaluation, two years after treatment was applied to the orchard (un presented results). Along our experiments, the question of chemical residues in the fruit was raised. We conducted a few residues analysis inspection. We did not find any residues in the fruit tested.

Hosen 2 l/h was registered for use for the control of mal secco in Israel in 2020 and is now recommended as part of the available treatments and widely used by growers for the control of the disease in young and newly planted lemon orchards as well as in older orchards.

We have found that treating lemon trees with "Hosen" (Futriafol 125 gr/liter, Adama, Israel) 2 liter/ hectare, five times a year, at October and November (one month apart), March (right after the first spring irrigation of the orchard), April and May (one month apart), by injection into the dripping system using a pump. Alongside strict sanitation of dry and symptomatic twigs and branches, while keeping the size of the trees to not more than 2.5 meters high, may control mal secco disease symptoms, reduce dramatically dry out of branches and death and uprooting of trees.

ACKNOWLEDGEMENTS

This work was supported by The Israeli Plant Production and Marketing Board, the Citrus division and by the Israeli chief scientist, ministry of agriculture and rural development as part of the "Mal secco "HOSEN" project.

STATEMENTS AND DECLARATIONS

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by David Ezra and Amos Ovadia. The first draft of the manuscript was written by David Ezra and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The authors declare no potential conflicts of interest.

Ethical Approval “not applicable”

Funding This work was supported by The Israeli Plant Production and Marketing Board, the Citrus division and by the Israeli chief scientist, ministry of agriculture and rural development as part of the "Mal secco "HOSEN" project.

Availability of data and materials “not applicable” all data and material relevant to this publication is presented in the manuscript.

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No competing interests reported.

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You are reading this latest preprint version

Control of Mal secco disease in lemon by drip irrigation of fungicide

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Abstract

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

Conclusions

Declarations

ACKNOWLEDGEMENTS

STATEMENTS AND DECLARATIONS

References

Additional Declarations

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