Threatening guest: Sclerotinia sclerotiorum in citrus

the fungus Sclerotinia sclerotiorum It is reported in hundreds of species of cultivated and weedy plants. Known as white mold, the disease caused by this fungus causes great damage to soybean, bean, cotton, sunflower, tomato, potato and lettuce crops, especially in regions or seasons with colder and more humid climates. The altitude of the region is also decisive for its emergence, prevailing in places higher than 700m and in areas irrigated by central pivot (Furlan, S.H., 2012).

    The main units of infection of the fungus (inoculum) are the resistance structures, the sclerotia, which remain viable for more than five years in the soil, and the ascospores, coming from the fruiting bodies (apothecia), which infect the flowers, an important source of energy for the first infections.

    In regions at higher altitudes there is a high chance of an epidemic occurring, resulting from lower night temperatures and long periods of dew. The problem becomes worse when rainfall is frequent at the flowering stage and beginning of fruiting, in synchrony with the closing of the sowing lines, which depends, above all, on the architecture, size, density and spacing of the plants.

    Symptoms appear throughout the aerial part of the crop, initially characterized by waterlogged lesions, which progress to rot and wilting. The presence of white cottony mycelium is easily observed on the surface of the soil and in plant tissues, where sclerotia subsequently form, internally or externally to the attacked parts.

    In citrus, the occurrence of S. sclerotiorum has been little reported around the world (Saharan & Mehta, 2008), for example, in Citrus volkameriana in Italy (Polizzi et al, 2011) and in Brazil, it is cataloged by Embrapa/Cenargen (http://pragawall.cenargen.embrapa.br/aiqweb/michtml/fgbd02a.asp), mentioning the Citrus sp. L. (Rutaceae) as one of the hosts, among many others. In this crop, it was described by Rosseti, V.V., in 2001, in the Illustrated Manual of Citrus Diseases. However, there is a lack of research work that scientifically proves its pathogenicity and damage to citrus fruits.

    The objective of this work was to report the occurrence of S. sclerotiorum as a pathogen for citrus crops in a cultivated area in Brazil and its pathogenicity confirmed by Koch's postulates, in addition to knowing the interactions between this citrus isolate and two others from soybeans and beans.

Laboratory experiment

 Stems and branches of citrus plants (Galego lemon), from an orchard in Castro, Paraná, approximately four years old, showing lesions and white mycelium in the tissues, at around 5% incidence, were collected in the winter/spring of 2011 and sent to the Phytopathology Laboratory of the Biological Institute, for diagnosis.

The standard procedure of direct and indirect isolation of part of the infected tissues was carried out, using Petri dishes containing PDA culture medium. After five days of incubation in BOD, at 22ºC, with a 12-hour photoperiod, colony growth began.

The fungus was picked and multiplied for subsequent inoculation in healthy citrus plants, of the same variety as the initial isolation. After the reproduction of symptoms, the lesions were re-isolated, in order to complete Koch's postulates and prove the diagnosis.

To study the interactions between the fungus isolates, cross-inoculation was carried out on three hosts: citrus, beans and soybeans, that is, an isolate from each crop was used, each of which was inoculated on different hosts. For this, citrus seedlings approximately 12 months old were used, coming from commercial nurseries, of five varieties: Galego, Pera, Dekopon, Tahiti and Ponkan, with beans from the Pérola cultivar and soybeans from the BRS 245 cultivar. legumes had 21 days of emergence on the day of inoculation.

The inoculation method adopted was mycelium colony discs measuring 0,8cm in diameter, grown in PDA for eight days, at a temperature of 22ºC and a 12-hour photoperiod. Five discs were placed per plant, secured with adhesive tape, using a total of five plants (replication) per pot for beans and soybeans, and five seedlings (replication) for citrus.

The inoculated plants were maintained in a growth chamber with nebulization, at a regulated temperature of 22ºC ± 1ºC and RH ≥ 70%, for ten days.

After four days of incubation, the first lesions began on soybean and bean leaves and, after seven days, they appeared on citrus leaves, when the first reading was carried out. The assessment was made based on the positivity or absence of symptoms on the inoculated leaves, that is, the presence or absence of leaf lesions.

After ten days of incubation, the severity of symptoms on the leaves was assessed, assigning grades from 1 to 6, based on the longest length of the lesions, where 1 = absence of lesions; 2 = lesions up to 1cm; 3 = 1,1cm to 2cm; 4 = 2,1cm to 3cm; 5 = 3,1cm to 4cm; 6 = above 5cm or complete leaf death.

Then, the fungus was re-isolated in each host, to confirm its pathogenicity, following the same methodology for isolating the fungus initially.

Confirmed presence

 Based on the symptoms obtained after inoculation and re-isolation of the studied fungus, the diagnosis of Sclerotinia sclerotiorum, the causal agent of white mold, on inoculated citrus leaves, reproducing the growth of typical colonies of the fungus with white mycelium and the presence of sclerotia. The incubation period of the disease in beans and soybeans was shorter than in citrus, that is, symptoms appeared four days after inoculation on bean and soybean leaves, and after another three days, in citrus, indicating that the tissues are less susceptible.

Positive reactions indicate the pathogenicity of the isolates or the susceptibility of the host species to the tested isolate, while negativity indicates the non-existence of pathogenicity, or, alternatively, the possibility of an escape, that is, the absence of resulting symptoms, for example , the lack of more suitable conditions for the development of the disease or any other unknown factor (Table 1).

Through cross inoculations of isolates from citrus, beans and soybeans, it was verified that: for isolates from beans and soybeans, symptoms were reproduced in both hosts, indicating that the bean isolate is pathogenic for soybeans and vice versa. For the citrus host, positivity was obtained when the citrus isolate was inoculated in the Galego, Tahiti, Dekopon and Ponkan varieties, which, with the exception of Pera, proved to be susceptible under the test conditions. The citrus isolate also reproduced symptoms in beans and soybeans. The bean isolate was pathogenic in citrus only for the Ponkan variety and the soybean isolate was not pathogenic in any of the citrus varieties tested (Table 1).

Due to the severity of the disease symptoms, it was observed that the Galego and Tahiti citrus varieties were the most susceptible to inoculation, with scores of 4,8 and 4,0, respectively, followed by the Dekopon variety, with a score of 2,7. The most resistant were Ponkan (grade 1,3) and Pera, which did not show symptoms when inoculated. It is noteworthy that the Pera orange was the only one that did not show a positive reaction when inoculated with any of the tested isolates (Table 2). This may indicate a better defense response of this variety to this pathogen, however, it does not exclude the possibility of escape and even a possible susceptibility reaction to another isolate of S. sclerotiorum not tested in this trial.

Comparing soybean and bean isolates, there does not seem to be any specificity between each of them and their respective hosts. This could be confirmed by the fact that the bean isolate showed similar aggressiveness towards both crops (severity scores of 4,5 and 4,6, respectively for bean and soybean plants). The soybean isolate was less aggressive than the bean isolate, for both crops, with scores of 3,3 and 3,5, respectively. Symptoms in soybeans, beans and citrus, after evaluations, evolved quickly, reaching 100%. The citrus isolate seemed to have greater specificity, as it caused greater severity of symptoms in citrus plants, especially in the Galician and Tahiti lemon varieties, in relation to the symptoms caused in beans and soybeans (Table 2).

To better conclude on the interactions between isolates and hosts, and evaluate their specificity, a greater number of isolates and varieties of each plant species should be used in crossings. In this work, despite the occurrence of some negative reactions, we believe in the possibility of escapes, which are common to this type of inoculation with mycelium discs used here due to their practicality and speed.

This work can confirm the pathogenicity of S. sclerotiorum in citrus plants, from an orchard located in a region with a cold climate. Damage studies on this crop are important due to the relevance of white mold on several other host plant species in Brazil.

 Table 1 - Positive (+) or negative (-) reaction of plants of five citrus varieties, one bean and one soybean, against cross-inoculations of isolates of S. slerotiorum, coming from their respective hosts, after seven days of incubation

Isolated from	Reaction of inoculated hosts
citrus	Bean	Soybean
Galego	Tahiti	Pear	Dekopon	ponkan	Pearl	BRS-245
Bean	-	-	-	-	+	+	+
Soybean	-	-	-	-	-	+	+
citrus	+	+	-	+	+	+	+

Isolated from

Reaction of inoculated hosts

citrus

Bean

Soybean

Galego

Tahiti

Pear

Dekopon

ponkan

Pearl

BRS-245

Bean

-

-

-

-

+

+

+

Soybean

-

-

-

-

-

+

+

citrus

+

+

-

+

+

+

+

 

 

Table 2 - Severity of symptoms (grades from 1 to 6, based on the longest length of the lesion, where 1 = absence of lesions and 6 = above 5cm) caused by S. sclerotiorum, in citrus, bean and soybean hosts, after ten days of cross inoculations

Isolated from	Severity of symptoms in inoculated hosts – notes
citrus	Bean	Soybean
Galego	Tahiti	Pear	Dekopon	ponkan	Pearl	BRS-245
Bean	1 BC	1 BC	BC 1	1 BC	3,6 to B	4,5 to A	4,6 to A
Soybean	1 bB	1 bB	1aB	1 bB	1 bB	3,3b A	3,5 bA
citrus	4,8 aA	4,0 aA	1BC	2,7 aB	1,3 BC	3,0 bB	1,6cc

Isolated from

Severity of symptoms in inoculated hosts – notes

citrus

Bean

Soybean

Galego

Tahiti

Pear

Dekopon

ponkan

Pearl

BRS-245

Bean

1 BC

1 BC

BC 1

1 BC

3,6 to B

4,5 to A

4,6 to A

Soybean

1 bB

1 bB

1aB

1 bB

1 bB

3,3b A

3,5 bA

citrus

4,8 aA

4,0 aA

1BC

2,7 aB

1,3 BC

3,0 bB

1,6cc

Averages followed by the same lowercase letter, vertically, and the same capital letter, horizontally, do not differ significantly from each other.

Click here to read the article in issue 78 of Cultivar Hortaliças e Frutas.