Crop Protection 130 (2020) 105073

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Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Management of common root rot and Fusarium foot rot of wheat using
Brassica carinata break crop green manure
Vito Campanella *, Claudio Mandala
�, Vincenzo Angileri, Claudia Miceli
Council for Agricultural Research and Economics, Research Centre for Plant Protection and Certification, V.le Regione Siciliana Sud-Est 8669, 90100, Palermo, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Brassica crop rotations have been associated with the reduction of pathogens, due to both the production of
Brassica carinata volatile sulfur compounds and changes in soil microbial community composition. In this research Brassica car­
Bipolaris sorokiniana inata, as break crop green manure, was evaluated for the control of common root rot and Fusarium foot rot of
Fusarium culmorum
durum wheat in a complete randomized experimental scheme. Field trials were performed on seven farms, in
Break crop
Biofumigation
some of the most representative areas of cereal production in Sicily, from 2011 to 2013. A total eradication of
Succeeding crop Bipolaris sorokiniana and Microdochium nivale populations following the B. carinata break crop, as compared to
wheat monoculture, was found in all experimental fields. Fusarium culmorum population reduction ranged be­
tween 90% and 66%, with an average of 82% considering all field trials data. Symptoms or signs of common root
rot were absent on wheat plants grown after the B. carinata break crop but were detectable on the wheat
monoculture. Both incidence and severity of Fusarium foot rot were also reduced, by an average of 40.6% and
56.3%, respectively, following the B. carinata break crop in six of the seven field trials. After B. carinata break
crop, wheat plant growth showed significant increases in height and weight, with an average increase of 48.3%
and 49.0% respectively. Durum wheat grain yield was also significantly greater following the Brassica green
manure, with increases ranging from 9.5% to 62.2%, as compared to wheat monoculture. B. carinata break crop
green manure was also characterized by interesting results in terms of disease management in comparison to
controls. These results suggest that the use of Brassicas such as B. carinata, as a break crop green manure, may
potentially provide significant reduction of pathogens and disease development as well as improvement in grain
yield of the succeeding crop.

1. Introduction consequently enhance the incidence and severity of diseases. Moreover,

Common root rot and Fusarium foot rot are the most widespread and
harmful wheat diseases worldwide (Hogg et al., 2010; Scherm et al.,
2013) and particularly in Italy where wheat is cultivated as principal
crop on 1,806.572 ha (Handiseni et al., 2017). The main species
responsible for disease include Bipolaris sorokiniana (Sacc.) Shoem,
Fusarium acuminatum (Ellis & Everhart), F. avenaceum (Fries) Saccardo,
F. equiseti (Corda) Saccardo, F. crookwellense (Burgess, Nelson & Tous­
soun), F. culmorum (W.G. Smith), Saccardo, and Microdochium nivale
(Samuel & Hallet), (Corazza and Santori, 2003; Campanella and Miceli,
2009), which are particularly harmful in warm areas and in water-stress
conditions (Cariddi and Catalano, 1990; Chekali et al., 2011). The latter
lead to greater susceptibility of the host plants, rather than an increase in
the virulence of the pathogens (Scherm et al., 2013). Wheat mono­
culture and short crop rotation may increase soil-borne pathogens and
some pathogenic fungi persist in soil as dormant, long-lived structures,
such as chlamydospores, which can survive several years without a host
plant (Scherm et al., 2013), or on the debris of previous crops (Bockus
et al., 2010). Several agronomic practices are carried out to control the
diseases, such as crop rotations with crops that are not intermediate
hosts of the pathogens (i.e. legumes, brassicas), or the use of healthy
seeds coated with fungicides and/or crop treatments (Ghosh et al.,
2018). However, due to inappropriate cultural practices and environ­
mental conditions favoring diseases, these approaches are often ineffi­
cient. Additional management strategies should be employed within an
integrated disease management approach, so as to achieve sustainable
production systems. Among alternative control methods, safer for both
humans and the environment, the use of effective crop rotations that are
selected for their ability to reduce soil-borne pathogens could be a valid
option. Brassica spp., used as break crop green manure, have been

* Corresponding author.
E-mail address: vito.campanella@crea.gov.it (V. Campanella).

https://doi.org/10.1016/j.cropro.2019.105073
Received 20 September 2019; Received in revised form 6 December 2019; Accepted 22 December 2019
Available online 24 December 2019
0261-2194/© 2020 Elsevier Ltd. All rights reserved.
V. Campanella et al.
associated with the reduction of many pathogens (Larkin and Griffin,
2007; Larkin, 2013; Srivastava and Ghatak, 2017). Different Brassicas
species are used as biofumigants because of their diverse capabilities to
produce sulfur compounds called glucosinolates, which in their native
form are devoid of any biological activity. Glucosinolates, in the pres­
ence of water and of the endogenous enzyme myrosinase, are hydro­
lyzed with the production of a series of breakdown products, mainly
isothiocyanates, organic cyanides, oxazolidinethiones, and thiocyanate
(Manici et al., 1997; Gimsing and Kirkegaard, 2009; Agerbirk and Olsen,
2012). These substances are responsible for the suppressive effects on
soil-borne pathogens, a phenomenon defined “Biofumigation” (Kirke­
gaard and Matthiessen, 2004). Furthermore, through the incorporation
of biomass into soil, significant amounts of carbon nutrients and other
substances are replenished, thus stimulating soil microbial activity
(Stark et al., 2008) by altering the physical, chemical, and biological
characteristics of soil (Larkin et al., 2010) thus contributing to pathogen
suppression (Cohen et al., 2005). In recent years, several authors have
highlighted the potential use of Brassicaceae and the related bio­
fumigation process to control numerous plant pathogens, such as
Gaeumannomyces graminis var. tritici (Angus et al., 1994; Kirkegaard
et al., 2000; Ennaıfar et al., 2005), Rhizoctonia solani, Phytophthora
erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Fusarium sam­
bucinum (Larkin and Griffin, 2007; Handiseni et al., 2017), Microdochium
nivale (Infantino et al., 2010; Ladhalakshmi et al., 2015), Pyrenochaeta
lycopersici, Verticillium albo-atrum (Giotis et al., 2009), and Verticillium
dahliae (Michel and Lazzeri, 2011). Among Brassica spp., Brassica car­
inata has shown noteworthy results both in the management of Phy­
tophthora cactorum and the increase of strawberry yield and fruit weight
(Barrau et al., 2009). B. carinata was also used to decrease the incidence
of P. capsici in pepper crops (Nunez-Zofio et al., 2011), reduce inoculum
density of B. sorokiniana and F. culmorum in wheat crop soils (Campa­
nella et al., 2013), as well as to lower inoculum density of P. cinnamomi
in Quercus cerris seedlings (Morales-Rodrigues et al., 2016). Further­
more, B. carinata exhibits both a total glucosinolate whose concentration
progressively increases until the flowering stage (Bellostas et al., 2004)
and higher glucosinolate concentrations in the roots system, in com­
parison to other Brassica species, among which B. nigra, B. rapa, and B.
juncea.
The objectives of this study were: i) to evaluate the use of B. carinata
break crop green manure to control pathogens responsible for, and
resulting in, the development of common root rot and Fusarium foot rot
of wheat;
ii) to assess the growth, development and yield of durum wheat
following B. carinata green manure compared to continuous wheat
monoculture.
2. Material and methods
2.1. Field sites and soil management
Field experiments were conducted over a 2-year period (2011–2013)
in 1.2 ha trial plots, on seven wheat farms (labelled by field codes A to
G), located in representative cereal-growing provinces in Sicily
(Table 1). Standard agronomic techniques were used, employing the
farmers’ own farming equipment. During the two-year research period,
soil tillage, involving soil inversion with a disc plough, to a depth of 30
cm, in the late summer, followed by secondary tillage with a disc har­
row, at the end of October, were carried out in order to prepare seed
beds. Sowing was performed by seeders for B. carinata (seed rate 8 kg
ha 1) and durum wheat (seed rate 240 kg ha 1) during the second weeks
of December and November, in the first and second year, respectively.
Brassica soil fertilization was performed with 150 kg/ha 1 of super­
phosphate (19%) at sowing time and 200 kg/ha 1 of ammonium nitrate
(27%) at the end of tillering, whereas for weed control, metazachlor
(431 g kg 1) was used at sowing and fluazifos-p-butile (134 g kg 1) at
stem elongation. Durum wheat soil fertilization relied on 300 kg/ha 1 of
2
Crop Protection 130 (2020) 105073
Table 1
Geographic coordinates and provinces of the different field sites involved in the
research.
Field code Site/Provinces Coordinates Altitude (meters a.s.l.)
A Vicari (PA) 37� 500 N, 13� 320 E 280
B Xirbi (CL) 37� 320 N, 14� 20 E 460
C Raddusa (EN) 37� 300 N, 14� 300 E 290
D Monreale (PA) 37� 530 N, 12� 570 E 190
E Ciminna (PA) 37� 530 N, 13� 310 E 570
F Marineo (PA) 37� 560 N, 13� 210 E 600
G Mussomeli (CL) 37� 290 N, 13� 490 E 340
Provinces of field sites: Palermo ¼ PA, Caltanissetta ¼ CL, Enna ¼ EN.
superphosphate at sowing and 200 kg/ha 1 of ammonium nitrate at the
end of tillering, while weed control made use of mefenpir-dietile (90 g
kg 1) iodosulfuron-metile-sodio (6 g kg 1), mesosulfuron-metile (30 g
kg 1), mixed with florasulam (5.8 g kg 1; 2.4 D 423 g kg 1).
During the first year, each trial plot was divided into two equal
subplots, one cultivated with Triticum durum (Desf.) cv. Simeto and the
other with Brassica carinata (A. Braun) cv. CT 180. For both years, three
2000 m2 test areas within each subplot were delineated and employed
for sampling. B. carinata was grown until the flowering phase (i.e. 61–65
BBCH scale, Biologische Bundesanstalt, Bundessortenamt and CHemical
industry) (Hack et al., 1992), then the vegetation was green manured
with a disc plough and immediately incorporated into the soil using a
disc harrow. Durum wheat was then grown until the end of the crop
cycle (June). Fields were left undisturbed until the next sowing bed
preparations (late summer). During the second year, durum wheat was
cultivated either on B. carinata or on wheat as the control, while test
areas were the same as in the first year.
2.2. Soil sampling and pathogen detection
Soil sampling was performed at pre-sowing in the first year to
quantify the presence of soil-borne pathogens. In the second-year, soil
samples were taken at tillering (29 BBCH scale) and at the flowering
phase (61–65 BBCH scale) of wheat, from both subplots. Samples were
collected at regular intervals along the two main diagonals, at no more
than 20 cm of depth from each test area via a 100-mm diameter probe,
after removing a superficial layer. From each test area, soil samples were
made up of five soil sub-samples, which were then pooled into a com­
posite sample. After collection, soil samples were left to dry at room
temperature for 7–10 days, then sieved (mesh with 2-mm openings) and
analyzed. The inoculum densities (ID) of soil-borne populations were
determined by a soil dilution plate method, using semi-selective agar
media:
– to isolate the total fungal population we adopted PDA (39 g/l; Oxoid
Ltd. Basingstoke UK); after autoclaving and cooling medium to 45 � C,
1.0 g streptomycin sulfate, 0.125 mg neomycin sulfate were added;
– we isolated Fusarium spp according to the following procedure: Nash
and Snyder (1962): PPA (peptone 15.0 g, KH2PO4 1.0 g, MgSO4 x 7
H2O 0.5 g, PCNB 750 mg, Agar 20.0 g, in 1L H2O; after autoclaving
and cooling medium to 45 � C, 1.0 g streptomycin sulfate, 0.125 mg
neomycin sulfate were added);
Finally, the procedure used to isolate for Bipolaris spp. was as follows:
– Dodman and Reike (1982): D-R (Soluble starch 10.0 g, NaNO3 3.0 g,
K2HPO4 1.0 g, MgSO4 500 mg, KCl 500 mg, FeSO4 x7H2O 50 mg,
agar 15.0 g, in 1L H2O; after autoclaving and cooling medium to 45
�
C, 100 mg streptomycin sulfate, 20 mg chlortetracycline HCl, 50 mg
kanamycin sulfate, 700 mg rosa Bengal, 10 mg benomyl, 1 mg cap­
tafol, 3 mg dichloran were added).
Ten grams of air-dried, 2-mm sieved soil were suspended in 90 ml of
V. Campanella et al.
sterile (0.05%) water agar (Leslie and Summerell, 2006) in 100 ml flasks
and maintained on magnetic stirrers for 3 min. While stirring, 10 ml of
this soil suspension were taken and diluted into 90 ml of sterile (0.05%)
water agar, thus obtaining a final dilution of 1:100. One ml of this soil
dilution was spread onto the PDA-, PPA- and D-R semi-selective media.
Ten (90-mm diameter) plates were arranged from each media and
incubated, in the dark, for 2 days at 20 � C. The soil suspension was then
rinsed under a slow stream of tap water and plates were again incubated
at 20 � C and exposed to 8 h of NUV lux (Sylvania 36W/BLB-T8) until the
appearance and visible colonies. For each 10-g soil sample, three rep­
licates were made as above. Soil moisture content was determined by
desiccating 20 g of sieved soil for 24/48 h at 104 � C. Every colony from
each semi-selective medium was counted and separately plated onto
PDA (39 g/l; Oxoid Ltd. Basingstoke UK) for identification. During
analysis of each soil sample, we counted and identified an average of
more than 30,000 colonies including saprophytes, weakly parasitic and
pathogenic fungi. We identified colonies belonging to 14 different
Fusarium species, but we particularly focused our attention on species
harmful to durum wheat, at the environmental and cropping conditions
found in Sicily. Moreover, all colonies resembling Fusarium spp. were
sub-cultured and identified as pure cultures on PDA and SNA (Spezieller
Nahrstoffarmer Agar) media (i.e. KH2PO4 1 g, KNO3 1 g, MgSO4*7H2O
0.5 g, KCL 0.5 g, Glucose 0.2 g, Sucrose 0.2 g, Agar 20 g in 1 L of distilled
water). 50-mm diameter Petri dishes were plated then incubated at 20
�
C � 1 � C for 7–14 days under NUV lux, alternating daylight (Philips TLD
36W/840) and darkness at 12-h cycles. Colonies were expressed as
number of colony forming units (CFUs) per gram of soil. Colonies were
identified by pigmentation, growth rate and by their microscopic fea­
tures as conidiogenus cells, micro/macroconidia and/or chlamydo­
spores, according to Sivanesan (1987) and Leslie and Summerell (2006).
All colonies belonging to the genus Fusarium were quantified, repre­
senting the total density of the inoculum of Fusarium spp. The results
were validated via PCR assays (data not shown), according to the CTAB
methods of Nicholson et al. (1996), then compared to international
isolate culture collections (American Type Culture Collection, ATCC
Manassas VA, USA). Data were reported for: i. the main soil-borne
pathogens at pre-sowing in the first-year; ii. the total soil-borne fungi;
iii. the total Fusarium population; and iv. the second-year data of the
main pathogens following the B. carinata break crop, relative to wheat
monoculture.
2.3. Wheat plant development, disease assessment and yield
Wheat plants from Brassica break crop and from wheat monoculture
were collected at the tillering phase (29 BBCH scale) to evaluate both
vegetative measures (height and weight), and incidence (I) and severity
(S) of the diseases. From each test area, 150 whole plants were harvested
at regular intervals along the two principal diagonals. Plants were rinsed
under tap water to remove soil debris, then were dried at room tem­
perature. Samples of each replicate of the two treatments were first
evaluated for incidence and severity of diseases, then measured for
height and fresh weight. The dry weight of whole plants was determined
after oven-drying at 104 � C until constant weight. Disease severity was
assessed using an empirical scale with values ranging from 0 to 4: 0 ¼
absence of symptoms/healthy; 1 ¼ slight browning at the base of the
culm/roots; 2 ¼ browning of approx. 50% of the culm/roots; 3 ¼ culm/
roots browning over 50%; 4 ¼ complete browning of culm/roots (Stack,
1992; Pasquini et al., 2003). Disease severity was evaluated according to
the Mckinney index: Σ [(v x n)/(N x V)] x 100, in which v ¼ numeric
value of the class; n ¼ number of observed cases for each class; N ¼ total
number of observed cases; V ¼ numerical value of the highest class.
Wheat yield was assessed by collecting plants from five randomly
selected 20-m2 areas in each of the 3 subplots subjected to the two
experimental treatments.
3
Crop Protection 130 (2020) 105073
2.4. Statistical analysis
All field trials had a completely randomized design. Within each trial
of both B. carinata and durum wheat crop rotations, three subplots were
identified and employed for all samplings in both years. All soil-borne
analyses were repeated twice and the data are expressed as the overall
average of the replicates.
In order to identify significant differences following B. carinata bio­
fumigation, all the data were verified for homogeneity and normality
and mean values were compared using the T Student test (p � 0.05).
Percentages were transformed into Bliss angular values (arcsine √%)
before analysis. Incidence and disease severity data are expressed as
percentages. Data are presented for each stage (by column) in Table 2,
whereas individual field trial results and means are shown (by row) in
Table 3 and subsequent tables.
3. Results
3.1. Soil sampling and pathogen detection
In aggregate, 14 Fusarium spp. were identified (i.e. F. acuminatum, F.
avenaceum, F. chlamydosporum, F. compactum, F. crookwellense,
F. culmorum, F. dimerum, F. equiseti, F. merismoides, F. oxysporum, F.
scirpi, F. semitectum, F. solani, F. sporotrichioides) as well as Microdochium
nivale. However, we focused only on the subset of Fusarium spp. most
harmful for durum wheat production, at the environmental and crop
conditions in Sicily (i.e. F. acuminatum, F. crookwellense, F. culmorum and
Microdochium nivale). The preliminary assessment of common root rot
and Fusarium foot rot of wheat show the constant presence of B. sor­
okiniana and F. culmorum in the seven field trials (Table 2). The average
inoculum densities of B. sorokiniana and F. culmorum were 256 and
1,380 CFUs per gram of soil, respectively. The greatest inoculum den­
sities of B. sorokiniana (540 CFUs) and of F. culmorum (3,014 CFUs) per
gram of soil were found in field trials C and D, respectively.
M. nivale was found only in three of the seven field codes and its
inoculum density was, on average, 61 CFUs per gram of soil; the highest
density (83 CFUs) per gram of soil was detected in field code D.
F. crookwellense was also detected in three field codes, with an
average inoculum density of 222 CFUs per gram of soil, while the peak
inoculum density was observed in field code A.
F. acuminatum was instead detected in four field codes, with an
average inoculum density of 387 CFUs per gram of soil, whereas field
code E showed the highest inoculum level (1,220 CFUs).
B. carinata break crop green manure significantly increased (p �
0.05) the total inoculum density of the soil-borne fungal population in
comparison to wheat monoculture at the tillering phase of wheat in field
codes A, B, D, E, and F (Table 3). No statistical difference was detected
between the two crop systems in field code C and only in field code G did
wheat monoculture show values of total population significantly higher
than Brassica break crop green manure.
As to the inoculum density of total soil-borne populations relative to
field codes A, B, C, D, and F there was no statistical difference between
the two crop rotation systems, at the flowering stage of wheat devel­
opment (Table 3). At this stage, B. carinata break crop green manure
significantly increased (p � 0.05) inoculum density of the total popu­
lation only in field codes E and G.
Overall, B. carinata break crop green manure significantly increased
(p � 0.05) total inoculum density of Fusarium spp. soil-borne population,
in comparison to wheat monoculture, both at tillering and flowering
stages of wheat plant development. In fact, values increased in six out of
seven field codes at tillering and in five at the flowering stage, with an
average increase of 33.3% and 29.3%, respectively (Table 4).
Wheat monoculture showed values of total inoculum density of
Fusarium spp. significantly greater than Brassica break crop green
manure only in field codes G and F, at the tillering and flowering stages
of wheat development, respectively.
V. Campanella et al. Crop Protection 130 (2020) 105073

Table 2
Inoculum density (ID) assessment of the main soil-borne pathogens at pre-sowing, during the first year.
Field code ID

B. sorokiniana (CFU g 1) F. acuminatum (CFU g 1

) F. crookwellense (CFU g 1
) F. culmorum (CFU g 1) M. nivale (CFU g 1
)

A 207 � 6.2 124 � 6.5 539 � 7.5 747 � 8.5 41 � 4.1

B 219 � 4.5 – – 1,053 � 7.9 –
C 540 � 3.8 – – 1,512 � 9.5 –
D 189 � 4.2 335 � 5.5 84 � 2.8 3,014 � 9.8 83 � 3.8
E 107 � 2.5 1,220 � 8.2 – 1,504 � 9.6 58 � 3.5
F 108 � 2.9 173 � 5.3 – 1,030 � 9.2 –
G 423 � 3.5 85 � 3.5 42 � 4.3 803 � 8.9 –

Mean 256 387 222 1,380 61

Each value represents the means of overall replicates � standard deviation. Data are compared within a row.
- Pathogen not detected.
Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C: Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo Province; F:
Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province.

Table 3 Table 5
Inoculum density (ID) of total soil-borne fungi observed in wheat grown after B. Inoculum density (ID) of the main soil-borne pathogens at tillering stage of
carinata break crop (Brassica) and after durum wheat monoculture (Wheat) at wheat development grown after B. carinata break crop (Brassica) and after
two different stages of wheat plant development. durum wheat monoculture (Wheat).
Field code ID ID Field Treatments ID (CFU g 1)
1 1 code
Tillering (CFU g ) Flowering (CFU g ) B. F. F. M.
sorokiniana acuminatum culmorum nivale
Brassica Wheat Brassica Wheat
A Brassica 0b 83 a 42 b 0b
A 28,156 a 19,433 b 25,933 a 21,900 a
Wheat 103 a 83 a 287 a 41 a
B 28,811 a 22,189 b 26,811 a 29,253 a
C 41,200 a 45,589 a 33,633 a 33,278 a B Brassica 0b – 43 b –
D 33,700 a 15,322 b 38,283 a 30,283 a Wheat 200 a – 430 a –
E 28,956 a 11,533 b 21,944 a 11,078 b
C Brassica 0b – 258 b –
F 45,256 a 14,755 b 17,056 a 12,811 a
Wheat 650 a – 1079 a –
G 20,200 b 32,500 a 19,678 a 15,356 b
D Brassica 0b – 83 b 0b
Data are presented for each stage and for each individual field code. Value Wheat 92 a – 1046 a 76 a
marked with the same letters are not statistically different (p � 0.05) according
to the Student T test. Data are compared within a row. E Brassica 0b 341 a 759 b 0b
Wheat 107 a 0b 2809 a 43 a
Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C:
Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo F Brassica 0b 173 a 44 a –
Province; F: Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province. Wheat 108 a 87 a 129 a –

G Brassica 0b 42 a 125 b –
Wheat 630 a 125 b 1834 a –
Table 4
Inoculum density (ID) of total Fusarium spp. soil-borne fungi in wheat grown Data were analyzed for each individual field code. Value marked with the same
after B. carinata break crop (Brassica) and after durum wheat monoculture letters are not statistically different (p � 0.05) according to the Student T test
(Wheat) at two different stages of wheat plant development. and. Data are compared within a row.
- Pathogen not detected.
Field code ID ID Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C:
Tillering (CFU g ) 1
Flowering (CFU g 1) Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo
Province; F: Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province.
Brassica Wheat Brassica Wheat

A 7,132 a 5,332 b 7,930 a 5,453 b

B 6,611 a 5,284 b 7,575 a 5,608 b
this pathogen registered a 66% decrease (Table 5).
C 6,139 a 5,611 b 6,757 a 4,383 b B. carinata increased (p � 0.05) inoculum density of F. acuminatum in
D 7,828 a 4,163 b 7,094 a 4,364 b field code E, while no statistical differences were observed in field codes
E 5,746 a 1,477 b 3,047 a 3,110 a A or F. In the latter, an increase of the inoculum density of about 50%
F 4,307 a 3,250 b 3,278 b 3,688 a
was observed. Inoculum density of F. acuminatum was reduced by 66%
G 3,676 b 6,753 a 3,948 a 3,338 b
following Brassica break crop green manure only in field code G. Given
Data are presented for each stage and for each individual field code. Value the substantial overlap in the inoculum densities of pathogens respon­
marked with the same letters are not statistically different (p � 0.05) according sible for common root rot and Fusarium foot rot of wheat at the flow­
to the Student T test. Data are compared within a row.
ering and tillering, only tables for the tillering stage are provided.
Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C:
Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo
Province; F: Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province.
3.2. Wheat plant development, disease assessment and yield

B. carinata break crop green manure decreased inoculum density of
B. sorokiniana, M. nivale and F. crookwellense (p � 0.05) with a complete
inhibition of these fungi in all field codes (Table 5). F. culmorum inoc­
ulum density also significantly lessened in almost all fields, with an
average drop of 82%. Statistically significant values were not reached,
however, for field code F in which, nevertheless, the inoculum density of
B. carinata increased (p � 0.05) plant height and weight in almost all
field trials in comparison to wheat monoculture (Table 6). The height of
wheat grown after B. carinata break crop green manure showed an
average increase of 48.3%, ranging from 64.5% (in field code C) and
18.6% (in field code E), while no statistical differences were found in
field code D. Besides the weight, the average height of wheat plants

4
V. Campanella et al. Crop Protection 130 (2020) 105073

Table 6 root rot was found in wheat plants from field code B. In general,
Vegetative parameters and productivity of wheat grown after B. carinata break compared to durum wheat monoculture, plants grown after B. carinata
crop (Brassica) and after durum wheat monoculture (Wheat). break crop showed an average relative reduction in incidence and
Field Treatments Height Fresh weight Dry weight Yield (t/ severity of 40.6% and 56.3%, respectively. In almost all field codes,
code (cm) (g) (g) ha) B. carinata break crop green manure significantly increased yield (p �
A Brassica 49.5 a 5.7 a 1.3 a 4.3 a 0.05) (Table 6). The production of wheat plants grown after B. carinata
Wheat 35.9 b 3.1 b 0.8 b 1.8 b break crop green manure showed an average increase of 52.3%, with the
B Brassica 45.5 a 6.1 a 1.4 a 4.6 a greatest yield value observed in field code C (62.2%). Despite an
Wheat 29.3 b 2.0 b 0.5 b 2.3 b increased yield of 9.3%, there were no statistical differences only in field
C Brassica 43.1 a 2.7 a 0.9 a 3.7 a
code D.
Wheat 26.2 b 1.2 b 0.3 b 1.4 b

D Brassica 45.2 a 4.4 a 1.4 a 2.1 a

4. Discussion
Wheat 44.9 a 4.3 a 0.9 b 1.9 a
In our investigation, inoculum density of the fungi populations was
E Brassica 37.6 a 3.1 a 0.8 a 1.8 a
Wheat 31.7 b 2.6 b 0.7 a 0.9 b significantly increased in the B. carinata break crop system compared to
wheat monoculture at the tillering stage of wheat development. Five of
F Brassica 31.4 a 1.9 a 0.4 a 3.4 a
Wheat 19.1 b 1.0 b 0.2 b 2.1 b
the seven field codes showed, in fact, an average increase of inoculum
density of the soil-borne fungi population of 47.2%. Soil enriched with
G Brassica 60.6 a 8.2 a 2.4 a 3.8 a
fresh plant material is known to increase the availability of substrates
Wheat 40.6 b 3.1 b 1.0 b 1.7 b
(Manici et al., 2004; Friberg et al., 2009; Nunez-Zofio et al., 2011) and
Data were analyzed for each individual field code. Value marked with the same can promote the increase of all saprophytic soil-borne fungi, among
letters are not statistically different (p � 0.05) according to the Student T test. which Fusaria (Leslie and Summerell, 2006). Therefore, the observed
Data are compared within a row.
increase in fungi may be linked to the additional organic substances in
Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C:
the soil, as highlighted by various authors. The latter have found that,
Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo
Province; F: Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province. after the addition of organic material to the soil, the microflora increases
significantly (Smolinska, 2000) and, in trials in which soils were
amended with either B. napus seed meal (Cohen et al., 2005), or crushed
grown after B. carinata showed an increase of 49.0%, with the greatest
Brassica spp. residues (Smolinska, 2000; Friberg et al., 2009; Srivastava
and the least increases found in field codes B (67.2%) and E (16.1%)
and Ghatak, 2017), the total soil-borne fungal load increased.
respectively. However, in field code D there were no statistical differ­
No statistical differences in fungi were found in field code C, whereas
ences. No symptoms or signs of common root rot were demonstrable at
the soil-borne fungal population increased in wheat monoculture rela­
the tillering stage of wheat after Brassica and wheat.
tive to B. carinata break crop green manure only in field code G. The
B. carinata break crop green manure also significantly (p � 0.05)
differences observed in fields C and G may be explained by a delayed and
reduced incidence of Fusarium root rot in A through C and E to G; its
incomplete incorporation of the biomass into the soil, reflecting some
severity was significantly reduced in field codes A, B, E, F and G
adverse environmental conditions encountered during green manure of
(Table 7), yet only in field code D was there no statistical difference
the B. carinata biomass. Nevertheless, there were no significant differ­
between the two crop systems, despite an observed reduction of 4.2%
ences to tabulate between the two crop systems at the flowering stage of
and 5.9% in incidence and disease severity, respectively. The greatest
wheat; in fact, data of soil-borne fungi populations were substantially
reduction (100%) both in terms of incidence and severity of Fusarium
similar to those of the tillering stage.
As reported by Smolinska and Kowalczyk (2014), our findings
Table 7 following B. carinata break crop, also showed an increase in Fusarium
Incidence (I) and severity (S) of Fusarium root rot of wheat grown after B. car­ spp. populations, except for field code G, at tillering, and F at the
inata break crop (Brassica) and after durum wheat monoculture (Wheat). flowering stage of wheat development, where the differences may have
Field code Treatments Fusarium root rot been due to shortcomings during the incorporation of the biomass into
the soil, as mentioned above.
I S
Although the inoculum densities of fungi were increased overall, in
A Brassica 38.3 b 16.8 b almost all field codes we observed a complete inhibition, i.e. no demo­
Wheat 59.6 a 35.8 a
strable inoculum densities, of some of the most harmful species for
B Brassica 0b 0b durum wheat cropping in Sicily, such as B. sorokiniana and M. nivale.
Wheat 21.2 a 13.6 a
Furthermore, a significant reduction of F. culmorum was also observed
C Brassica 47.5 b 25.3 a following the B. carinata break crop green manure.
Wheat 66.1 a 27.9 a Zasada et al. (2003) reported that susceptibility of Fusarium spp. to
D Brassica 73.6 a 36.1 a brassicaceous amendments varies greatly. This could also be the case for
Wheat 76.8 a 38.4 a F. acuminatum, which in our trials seemed not to be affected by the
E Brassica 12.9 b 6.0 b suppressive effects of biofumigation. Nevertheless, we highlight the
Wheat 21.9 a 11.8 a observation in this study inasmuch as F. acuminatum is known to
F Brassica 48.7 b 20.2 b determine significant reductions of germination and vegetive vigor of
Wheat 65.4 a 33.9 a durum wheat seedlings (data not shown), as reported by Hill and Blunt
G Brassica 43.3 b 18.1 b (1994) and Mergoum et al. (1998). Conversely, F. crookwellense proves
Wheat 48.8 a 29.7 a to be susceptible to brassicaceous amendments. In fact, it could not
isolated after the B. carinata break crop green manure and, on a par with
Data were analyzed for each individual field code. Value marked with the same
letters are not statistically different (p � 0.05) according to the Student T test. F. acuminatum, is equally harmful for germination of durum wheat (data
Data are compared within a row. not shown), besides being pathogenic to wheat crops, according to re­
Field codes: A: Vicari, Palermo Province; B: Xirbi, Caltanissetta Province; C: ports by Chehri et al. (2011).
Raddusa, Enna Province; D: Monreale, Palermo Province; E: Ciminna, Palermo In our trials. B. carinata was chopped, then incorporated into the soil
Province; F: Marineo, Palermo Province; G: Mussomeli, Caltanissetta Province. at the flowering stage, so all the biomass contributed to the enrichment

5
V. Campanella et al.
of soil glucosinolates. These features most certainly contributed to the
suppressive effects on pathogens responsible for common root rot and
Fusarium foot rot of wheat. In research that used B. carinata to manage
Phytophtora cactorum of strawberry (Barrau et al., 2009), P. capsici of
pepper (Nunez-Zofio et al., 2011) B. sorokiniana and F. culmorum of
wheat (Campanella et al., 2013) P. cinnamomi of Quercus cerris (Moral­
es-Rodrigues et al., 2016), the authors found similar reductions of
soil-borne pathogens to those found in our trials. The mechanisms
whereby B. carinata exerts its antagonistic activity on pathogens
responsible for common and Fusarium foot rot of wheat are undoubtedly
linked to multiple factors, such as: i. The interruption of the
host-pathogen cycle. This condition, of course, may reduce the pro­
duction and survival of inoculum, although Fusarium spp. can survive as
soil saprophytes for many years, even in the absence of a host (Scherm
et al., 2013); ii. Direct pathogen inhibition due to the release of iso­
thiocyanates from the hydrolysis of glucosinolates, well demonstrated
both in vitro (Angus et al., 1994; Manici et al., 1997; Larkin and Griffin,
2007., Handiseni et al., 2017), and in vivo (Subbarao et al., 2007; Porras
et al., 2008; Giotis et al., 2009; Larkin et al., 2010); iii. The amount of
glucosinolate compounds produced both by roots and B. carinata tissues.
In B. carinata, total glucosinolate concentrations increase until the
flowering stage and, in the roots system, higher glucosinolate concen­
trations have been observed, in comparison to B. juncea, B. nigra and B.
rapa (Bellostas et al., 2004).
In our trials, a statistically significant increase in both height and
weight of durum wheat plants following the B. carinata break crop green
manure was observed. Beneficial effects comparable to ours have been
reported in tomato plants grown in soil amended with B. carinata for the
control of Meloidogyne incognita (Mocali et al., 2015). Similar results
were reported in apple orchard soils amended with B. napus seed meal
for the control of Rhizoctonia solani (Cohen et al., 2005), as well as in soil
amended with B. oleracea for the control of Verticillium wilt in strawberry
plants (Subbarao et al., 2007). The improvement of soil properties due to
B. carinata break crop green manure may be responsible for the increases
in the vegetative parameters observed in our trials. These conclusions
are also corroborated by several authors who added fresh organic matter
to soil to produce physical, chemical (Stark et al., 2008), biological
(Larkin et al., 2011) and pedological conditions that render soils less
hospitable for many pathogens (N’ Dayegamiye and Tran, 2001). These
authors observed that the greatest increases in soil carbon and nitrogen
content occurred in soils receiving B. hirta and B. campestris green
manure, compared to controls devoid of green manure applications. The
improved soil conditions and the suppressive effect of B. carinata may
have induced a reduction of both incidence and severity of common root
rot and Fusarium foot rot. No symptoms or sign of common root rot were
detected in wheat plants grown after B. carinata break crops. B. sor­
okiniana was isolated in all field trials during the preliminary assessment
during the first year of investigation, with an average value of 244 CFUs
per gram of soil. Analogously, F. culmorum was found in all field trials
with an average value of 1,380 CFUs per gram of soil. After B. carinata
break crop green manure, the inoculum density of B. sorokiniana was not
detected in any field trial and those of F. culmorum was, on average, 193
CFUs per gram of soil.
Incidence and severity data for Fusarium foot rot showed a signifi­
cant reduction in six and in five field trials, respectively. Similar results
were found in a study in which B. carinata was used as the organic
amendment to manage P. capsici in pepper crops (Nunez-Zofio et al.,
2011). Results similar to ours were obtained with other Brassicas used
for soil amendments to control Corky root rot and Verticillium wilt of
tomato (Giotis et al., 2009), or to manage Phytophthora blight on squash
(Ji et al., 2012) and still again in a study on the effects of B. napus break
crop to control soil-borne potato diseases (Larkin et al., 2010). No sta­
tistical differences were found for severity of Fusarium foot rot in field
code C, nor for incidence or severity in field code D. The latter results
seem difficult to interpret at present. Generally, the higher the inoculum
density of the pathogen in the soil, the higher the disease burden
6
Crop Protection 130 (2020) 105073
(Ippolito et al., 1991). Therefore, these results could depend on the
higher inoculum levels of F. culmorum, of about 3,000 CFUs per gram of
soil, approximately double the average values of all the other field
codes.
Durum wheat yield was also significantly higher in wheat plants
cultivated after B. carinata break crop green manure in six of the seven
field codes, with an average increase of 51.8%. Compared to the control
crops, the addition of fresh organic matter accruing from Brassica green
manure provides an increased availability of mineral elements, as well
as by the degree of N mineralization and an improvement in soil prop­
erties, with significant effects on wheat yield (N’ Dayegamiye and Tran,
2001; Srivastava and Ghatak, 2017). In our trials, B. carinata green
manure was performed during the flowering phase of Brassica, at the
end of spring (May), while wheat sowing was performed in late autumn
(November). So it is plausible to assume that mineralization, occurring
throughout this period, made more nitrogen available for wheat de­
mands thus increasing yield. In research in which incorporation of B.
carinata was also combined with soil solarization (Barrau et al., 2009)
the authors found an increase in strawberry yield; similarly, Subbarao
et al. (2007) demonstrated that rotations with broccoli and Brussels
sprouts (B. oleracea), followed by the postharvest incorporation of the
respective residues, increased fruit yield of strawberry. Larkin et al.
(2010) also reported a significant increase in tuber yield subsequently to
crop rotation with canola (B. napus), as compared to continuous potato
cultivation.
5. Conclusion
The results of our investigation show the effectiveness of B. carinata
break crop green manure as a means to control of soil-borne pathogens
responsible for common root rot and Fusarium root rot of wheat. The
study confirms that the sensitivities of soil-borne pathogens to bio­
fumigation differ. In particular, B. sorokiniana, responsible for common
root rot, is highly sensitive. Our findings also highlight a significant
reduction in inoculum densities, incidence and severity of the subset of
Fusarium spp. most harmful for durum wheat production, at the envi­
ronmental and crop conditions in Sicily. Furthermore, we obtained
remarkable results in the increase of wheat yields. Our findings seem to
indicate that B. carinata break crop green manure might, however, be
less effective when the inoculum density of F. culmorum is high, as
observed in field code D. Nevertheless, further research is needed to
confirm the effects of Brassica break crop green manure in other wheat
cultivation contexts having different levels of inoculum densities of soil-
borne pathogenic fungi. Therefore, B. carinata break crop green manure
should be promoted in rotation with durum wheat in Mediterranean
areas for its action both in controlling pathogens and/or diseases and for
its capacity to improve the production of succeeding wheat crops.
Moreover, B. carinata could represent an essential factor of an integrated
and environmentally-friendly disease-management program.
Author contribution
Vito Campanella: choice and field design, soil management, soil
sampling, pathogens detection and identification, disease assessment,
yield assessment, Formal analysis, statistical data analysis, writing
manuscript, Claudio Mandala: soil management, soil sampling, media
preparation, disease assessment, yield assessment, Vincenzo Angileri:
soil management, soil sampling, media preparation, disease assessment,
yield assessment, Claudia Miceli: choice and field design, statistical data
analysis, writing manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
V. Campanella et al.
Acknowledgements
This research was financed by the project “Energie rinnovabili da
colture Agricole: progetto di cooperazione per l’innovazione e lo svi­
luppo tecnologico del settore agroenergetico in Sicilia”, funded by
“Misura 124-PSR Sicilia 2007–2013” della Regione Siciliana.
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